bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential development of helper and suppressor functions, antibody titers and functional avidities to a streptococcal antigen in rhesus monkeys
Authors: Lehner Thomas , Caldwell Jill , Avery Janet
Published: February 2007

Sequential development of antibody titer, functional avidity, helper and suppressor activities were investigated in rhesus monkeys. These were immunized with a single dose of 0.1 μg to 10 mg of a streptococcal protein antigen (SA) in aluminium hydroxide. The IgG antibody titers followed the classical pattern first established in mice, of high‐dose and low‐dose tolerance with intermediate doses of immunity. This was correlated with a similar pattern of functional avidity of IgG antibodies, as measured by a dissociation assay. Helper and suppressor functions were assayed in parallel by inducing the corresponding factors from monkey lymphocytes in Marbrook flasks and testing the factors which cross the species barrier in cooperative cultures with CBA mouse spleen B cells. A progressive modulation of helper and suppressor activities was elicited by the increasing doses of SA, during the initial 28 days after immunization. Thus, dominant suppressor with minimal helper activity, IgG antibody titer and functional avidity were elicited by 0.1 μg SA. However, 1 or 10 μg SA induced dominant helper with minimal or transient suppressor activity and high IgG antibody titers and functional avidity. Somewhat intermediate responses were elicited by 100 μg SA, but 1 mg and especially 10 mg SA induced dominant suppressor and minimal helper activity, with low IgG antibody titers and functional avidities. When the immune response was established, about 28 days after immunization, the intermediate dose of SA elicited IgG antibodies with high titer and functional avidity, high T cell helper but low suppressor activities. In contrast, both high‐ and low‐dose SA induced partial tolerance, with low IgG antibody titer, functional avidity and T cell helper activity. These studies suggest cyclical development of helper and suppressor functions during the 4 weeks after immunization. The emergence of a dominant helper or suppressor function is antigen dose dependent.
Immunoregulation of lysozyme‐specific suppression. I. Induction and suppression of delayed‐type hypersensitivity to hen egg‐white lysozyme
Authors: Colizzi Vittorio , Doria Gino , Adorini Luciano
Published: February 2007

Subcutaneous immunization with hen egg‐white lysozyme (HEL) in complete Freund's adjuvant induces, both in antibody responder and nonresponder mice, a classical delayed‐type hypersensitivity (DTH) reaction evaluated as footpad swelling. This response can be specifically transferred to naive recipients by Lyt‐1+2− T cells and passive transfer is restricted by genes mapping in or to the left of the I‐A region of the H‐2 complex.

Fine antigenic specificity analysis shows that HEL‐primed T cells mediating DTH recognize ring‐necked pheasant egg‐white lysozyme, a lysozyme closely related to HEL, but fail to respond to human lysozyme, differing from HEL at 40% amino acid residues. Complete cross‐reactivity between native and denaturated (reduced and carboxymethylated) HEL is exhibited by T cells involved in the DTH response. Subcutaneous injection of HEL coupled to spleen cells is also able to induce antigen‐specific and genetically restricted DTH responses whereas the same cells administered by i.v. or i.p. route induce predominantly suppressor T cell activation. These suppressor T cells specifically inhibit the induction phase of DTH reactivity to HEL.
Frequencies of phosphorylcholine‐specific and T15‐associated 10/13 idiotope‐positive B cells within lipopolysaccharide‐reactive B cells of adult BALB/c mice
Authors: Lévy Martine
Published: February 2007

The frequencies of lipopolysaccharide‐reactive B lymphocytes in adult BALB/c spleen cells were determined by limiting dilution experiments to be between 1 in 50 and 1 in 100. Within this reactive population, the frequencies of B lymphocytes specific for phosphorylcholine (PC) and/or binding a TEPC 15‐associated idiotope‐specific monoclonal antibody (10/13) were found to be between 1 in 50 and 1 in 500. Binding of these antibodies to immobilized PC or to monoclonal idiotope‐specific antibodies could be inhibited by soluble PC in a solid‐phase radioimmunoassay. Around half the PC‐specific B cell clones were found to also bind 10/13 idiotope‐specific monoclonal antibody, while the rest were either PC specific, but idiotope‐negative or idiotope‐positive, and unable to bind immobilized PC.
Comparison of thymic and peripheral T cell Ly‐2/3 antigens
Authors: Walker Ian D. , Murray Brendan J. , Hogarth P. Mark , Kelso Anne , McKenzie Ian F. C.
Published: February 2007

Major structural differences occur between the thymic and peripheral T cell forms of the Ly‐2/3 antigen. Thymus Ly‐2/3 consists of similar amounts of two types of disulfide‐linked heterodimer, αβ and α′β (Mra = 38000, Mra′ = 35000, Mrβ = 30000). In contrast material from peripheral T cells consists almost exclusively of αβ dimers. The α chains of thymus and peripheral T cells differ also in isoelectric point with the thymic α chain being the more acidic. Based on peptide mapping experiments the α and α′ chains of thymus are likely to be alternatively modified forms of the same polypeptide backbone.

Individual T cell clones or T cell tumors propagated in vitro exhibit either a typical thymus or a typical peripheral T cell Ly‐2/3 polypeptide pattern indicating that the synthesis of both α and α′ chains can occur in the same cell. The heterogeneity of thymic Ly‐2/3 can be considerably reduced by removal of sialic acid residues, and after desialylation the α chains of thymus and a cloned cytotoxic T lymphocyte (CTL) line cannot be electrophoretically distinguished. If Ly‐2 structures affected the antigen specificity of CTL, a different structural variant would be expected in individual clones. The electrophoretic identity of desialylated thymus and CTL α chains suggests that Ly‐2 does not exhibit clonal variation in polypeptide structure and, therefore, cannot contribute to antigen specificity.
Synergistic protection by specific antibodies and interferon against infection by Trypanosoma cruzi in vitro
Authors: Plata Fernando , Wietzerbin Juana , Pons Francisco Garcia , Falcoff Ernesto , Eisen Harvey
Published: February 2007

Trypanosoma cruzi infects many mammalian cell types in vitro, including fibroblasts and macrophages. We have analyzed and compared the infection of mouse 3T3 fibroblasts and J774 macrophage‐like tumor cells by cloned Y‐A1.2 T. cruzi trypomastigotes. The effects of T. cruzi‐specific antibodies and of interferon (IFN) on infection were considered. Specific antibodies protected 3T3 fibroblasts from infection by T. cruzi, but increased the relative infectivity for J774 cells due to binding of the opsonized parasite to J774 Fc receptors. It was also apparent that IFN‐α,β (produced by fibroblasts) and IFN‐γ (produced by T lymphocytes) activated trypanocidal activities in both 3T3 and J774 cells, although IFN‐γ was 100 to 2000 times more effective than IFN‐α,β on the basis of IFN antiviral units added per culture. When anti‐T. cruzi antibodies and IFN‐α,β or IFN‐γ were used jointly, a synergistic protective effect was observed with both 3T3 fibroblasts and J774 cells. These results suggest that B and T lymphocytes might collaborate in vivo in the protection against T. cruzi infections by the production of anti‐trypomastigote antibodies and IFN‐γ, respectively.
Secretion of a macrophage‐activating factor distinct from interferon‐γ by human T cell clones
Authors: Andrew Peter W. , Rees Ann D. M. , Scoging Anne , Dobson Nicola , Matthews Ruth , Whittall J. Trevor , Coates Anthony R. M. , Lowrie Douglas B.
Published: February 2007

Supernatants from clones of human T lymphocytes that were responding to a purified Mycobacterium tuberculosis antigen were able to activate macrophages and macrophage‐like myeloma cells (U937) to release increased amounts of the microbicidal agent hydrogen peroxide. The activity was not neutralized by monoclonal antibody against interferon‐gamma (IFN‐γ), was greater than could be accounted for by the IFN‐γ activity in the supernatants, and was separated from IFN‐γ by high performance liquid chromatography. It is evident that IFN‐γ is not the only macrophage activator released by T lymphocytes responding to microbial antigen, and may not even be the main one to enhance antimicrobial activity in infections such as tuberculosis.
IgG pair formation on one antigenic molecule is the main mechanism of synergy between antibodies in complement‐mediated lysis
Authors: Hughes‐Jones Nevin C. , Gorick Barbara D. , Miller Nigel G. A. , Howard Jonathan C.
Published: February 2007

Two rat IgG2b monoclonal antibodies bound to different epitopes on a single RT1Aa antigen molecule synergize in their induction of complement‐mediated lysis of the red cells. The mechanism of this synergistic action has been investigated by determining the C1q: IgG relationship on red cells using the fluorescence‐activated cell sorter and making use of the considerable variation in antigen density between individual red cells. With a synergistic pair of antibodies, the C1q: IgG ratio was approximately 1 and independent of antibody density per cell, indicating that the antibody molecules are present as closely associated pairs, each pair binding two C1q molecules and thus forming a cyclic tetramer. When one of the antibodies was used alone, the number of C1q molecules bound relative to the amount of antibody was higher than expected from theoretical considerations based on a presumed random distribution of antigen‐antibody complex. This result can be explained if there is some mobility of the complex within the membrane. The interpretation of the results gives strong support to the hypothesis that C1q must bind bivalently to rat IgG2b complexes.
Clonal endurance and clonal burst size: novel indices of helper T cell heterogeneity
Authors: Miller Richard A.
Published: February 2007

This report presents a method for examining the behavior of individual antigen‐responsive helper T cell precursors (pHTL), following their repeated encounter with antigen. In our model system, lymph node cells from mice immunized with keyhole limpet hemocyanin (KLH) are cultured in vitro under limiting dilution conditions, using responder cell doses chosen to ensure that each culture well contains, at the outset, at most a single KLH‐reactive helper T cell precursor. Those wells which in fact did receive a KLH‐reactive pHTL are identified by the accumulation of the helper T cell product, interleukin 3 (IL 3), during a 6‐day culture interval. The lymphocytes generated during this culture interval are washed, divided into replicate aliquots, and then further cultured (with and without additional KLH) to see if the original pHTL has generated cells which are capable of further lymphokine production.

Using this approach, we found a surprising amount of variation from clone to clone in two measurements. Clones differed greatly in “endurance”, that is in the ability of progeny cells to continue to secrete IL 3 in the absence of additional antigenic stimulation. Among 148 clones tested, 90 were found not to continue lymphokine secretion in antigen‐free secondary cultures, while IL 3 production among the other 58 clones varied from 1 to 128 arbitrary units. Individual clones also varied greatly with respect to their ability to secrete additional IL 3 in response to additional antigen, and in “burst size”, that is in the number of new, antigen‐responsive helper cell precursors which they can generate during the initial culture interval; 43% of the clones produced no new pHTL, while others produced as many as 800 new pHTL cells in an 8‐day period. Neither measure correlates strongly with the amount of lymphokine originally produced by the initial “parental” pHTL.

This method will allow us to examine the factors which influence the allocation of clonal progeny cells into lymphokine producing effectors and antigen‐sensitive “memory” T cells.
Cell surface events which may initiate lysosomal enzyme secretion by human monocytes
Authors: Leoni Patricia , Dean Roger T.
Published: February 2007

Freshly isolated and subsequently matured human monocytes secreted lysosomal hexosaminidase in response to exposure to IgG‐Sepharose, but not certain derivatized control Sepharoses. The cells bound selectively to the surface of IgG‐Sepharose (and not the control Sepharoses) but because of the large size of the particles, could not ingest them. Since the soluble IgG was covalently linked to the Sepharose and free soluble IgG was not an inducer of secretion, the secretion was thus induced directly at the cell surface. Zymosan, a yeast cell wall particle which contains a mannan, was also able to induce secretion in the various monocyte stages under study. It could even bind to the cell surface of fresh monocytes which lacked the receptor for mannose‐terminated glycoproteins, and induce secretion in these cells. The mannose receptor appeared as monocytes matured, and the Ir number on the surface was increased by the action of lymphokines. Although zymosan‐induced secretion could be inhibited by mannose and certain other sugars, these seemed to have some complex metabolic effects in human monocytes (which previous work with mouse macrophages has not revealed). Thus, it was not possible to demonstrate whether zymosan could initiate secretion directly by interaction at the monocyte surface mannose glycoprotein receptor.
Helper function of human T cells with different affinity for Helix pomatia A hemagglutinin in a tetanus toxoidinduced B cell differentiation system
Authors: Wikén Margareta , Hellström Ulla , Perlmann Peter
Published: February 2007

T cells from human peripheral blood were enriched in T4+ cells by lysis of T8+ cells with the monoclonal antibody OKT8 plus complement. The T4+ subset was separated into 4 fractions differing in avidity for the lectin Helix pomatia A hemagglutinin (HP). The fractions were studied for their capacity to help autologous B cells to differentiate and mature into immunoglobulin (Ig) synthesis and secretion after activation with tetanus toxoid (TT) in vitro. To ensure the antigen specificity of induction, very low doses of TT (1–100 ng/ml) were used for activation of the lymphocytes, all obtained from previously sensitized donors. The T4+ cells with low avidity for HP (fractions HP‐I and HP‐II) exerted little help for B cell differentiation. Removal of these cells enhanced the helper function of the remaining T4+ cells, indicating that fractions HP‐I and HP‐II contained suppressor cells. In contrast, efficient B cell help was provided by T4+ cells with high avidity for HP (fractions HP‐III and HP‐IV). However, these fractions differed in the quality of help provided. Thus, while HP‐III cells induced IgG secretion, HP‐IV cells mainly induced IgM secretion. Moreover, while the Ig secreted after help from HP‐III cells was TT‐specific antibody, the Ig secreted by B cells in the presence of autologous HP‐IV cells was polyclonal, probably reflecting induction of B cells, differing in their responsiveness to signals provided by different types of T cells. The results indicate that T4+ cells vary in their stage of differentiation as seen by differences in expression of the HP marker and that differences in HP‐marker expression appear to be associated with differences in cellular functions.
Effects of μ‐specific antibodies on B cell growth and maturation
Authors: Leanderson Tomas , Forni Luciana
Published: February 2007

Over a wide range of concentrations affinity‐purified rabbit anti‐mouse μ chain antibodies, or their F(ab′)2 fragments, inhibit the appearance of immunoglobulin‐secreting cells (plaque‐forming cells; PFC) in lipopolysaccharide‐stimulated murine spleen cell cultures without affecting proliferation. Both IgM and IgG PFC are inhibited although the number of blasts bearing surface IgG remains unaltered. The IgM and IgG PFC response could be reconstituted to normal levels in cell cultures suppressed by μ‐specific antibodies by the addition of supernatants from in vitro propagated helper T cell clones, or from EL4 lymphoma cells induced with phorbol ester. Interleukin 1‐containing P388 supernatant, or recombinant DNA‐derived murine interferon‐γ, did not reconstitute the PFC response in cell cultures suppressed by μ‐specific antibodies, indicating that other factors are responsible for these effects. When spleen cell cultures, pre‐activated with either lipopolysaccharide or monoclonal mouse μ‐specific antibodies coupled to Sepharose, were exposed to EL4 supernatants in the presence of soluble μ‐specific antibodies, maturation to secretion was inhibited while proliferation was not. The implications of these findings on assay systems for B cell growth and maturation factors are discussed.
Frequency analysis of cytolytic T lymphocyte precursors (CTL‐P) generated in vivo during lethal rabies infection of mice. II. Rabies virus genus specificity of CTL‐P
Authors: Reddehase Matthias J. , Cox James H. , Koszinowski Ulrich H.
Published: February 2007

Cytolytic T lymphocyte precursors (CTL‐P) were sensitized in vivo by intraplantar infection of C57BL/6 mice with a lethal dose of rabies virus, strain ERA (ERA). As a result of sensitization CTL‐P matured to interleukin‐receptive CTL‐P (IL‐CTL‐P) that could be expanded in vitro to Thy‐1+, Lyt‐2+ CTL clones in the presence of IL without subjection to antigen‐driven selection. After infection with ERA, IL‐CTL‐P‐derived CTL lysed fibroblasts infected with rabies virus but not those infected with another rhabdovirus, the vesicular stomatitis virus. These CTL, however, did not discriminate between fibroblasts infected with the serologically closely related laboratory strains of classic rabies virus, ERA and HEP‐Flury, and the serologically distinct rabies‐related African isolate Mokola. This finding implies that in vivo sensitized IL‐CTL‐P recognize common genus‐specific determinants expressed on cells infected with members of the lyssavirus genus.
Macrophage antitumor activity: impaired responsiveness to interferon‐γ of macrophages from genetically defective mice
Authors: Boraschi Diana , Censini Stefano , Tagliabue Aldo
Published: February 2007

Macrophages (Mϕ) from the genetically defective mouse strains C3H/HeJ, A/J and P/J were unable to develop high levels of antitumor activities when stimulated either with immune recombinant interferon gamma (IFN‐γ) or with nonimmune IFN‐α and IFN‐β, as compared to Mϕ or normal C3H/HeN mice. IFN‐γ appeared to be an exceptionally good activator of C3H/HeN Mϕ, as it could induce tumoricidal capacities 3000 times more efficiently than nonimmune IFN. The high efficiency of IFN‐γ as Mϕ activator was indeed confirmed on defective Mϕ. In fact, at high doses IFN‐γ could also induce significant levels of both cytolytic and cytostatic activity in Mϕ of A/J and P/J mice, although it could not increase cytotoxic activities of C3H/HeJ Mϕ.
Beneficial effect of cyclosporin A on the lymphocytic choriomeningitis virus infection in mice
Authors: Saron Marie‐Françoise , Shidani Babak , Guillon Jean‐Claude , Truffa‐Bachi Paolo
Published: February 2007

The effect of cyclosporin A on the course of lymphocytic choriomeningitis virus infection in mice was investigated. Evidence is presented that administration of this immunosuppressive drug spares a majority of lethally infected mice. This beneficial effect is different from the one obtained with other treatments leading to the abolition of T cell functions. Surviving animals rapidly eliminate the virus and produce high titers of neutralizing IgG antibodies.
Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor
Authors: Fischer Elizabeth , Kazatchkine Michel D. , Mecarelli‐Halbwachs Lise
Published: February 2007

Formation and function of the classical (C4b, 2a) and alternative (C3b, Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b, 2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b, 2a(C4Nef)‐bearing cells in a dose‐dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell‐bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b, Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b, Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.
Interleukin 2 and interferon‐γ are not sufficient to induce natural killer‐like activity in human T cell clones
Authors: López‐Botet Miguel , Toribio Maria Luisa , Moretta Lorenzo , De Landázuri Manuel O.
Published: February 2007

The role of interleukin 2 (IL2) and interferon (IFN)‐γ in the maintenance of natural killer (NK)‐like activity mediated by human cytolytic T cell clones was investigated. When cultured in standard supernatant (SN) obtained by phytohemagglutinin stimulation of human mononuclear cells (containing both IL2 and IFN activities), several mixed lymphocyte culture‐derived clones displayed cytolytic activity directed against the K562 cell line; in addition, some of these clones also lysed specific target cells bearing the sensitizing alloantigens. When cultured in IL 2‐containing SN obtained from the Jurkat cell line, the NK‐like activity was selectively abrogated within 4–5 days. However, by culturing cells again in standard SN the cytolytic activity was completely restored. The possibility that inhibitory molecules in Jurkat SN could be responsible for the loss of NK‐like activity was ruled out by experiments showing that cytotoxicity could be induced by mixtures of Jurkat and standard SN. In addition, a source of affinity chromatography‐purified IL2 also failed to maintain NK‐like activity. Moreover, addition of recombinant IFN‐γ in combination with purified IL2 did not prevent the loss of NK‐like activity. The present results indicate that neither IL2 nor IFN‐γ are sufficient to maintain NK‐like activity and suggest that other molecule(s) are required.
Selective and polyclonal induction of high levels of λ light chain‐bearing immunoglobulins in BALB/c and SJL mice
Authors: Primi Daniele , Sanchez Pierre , Cazenave Pierre‐André
Published: February 2007

Polyclonal B cell activation is V gene independent and consequently results in the unselected expression of both Vλ and Vϰ genes. In an attempt to selectively and polyclonally trigger λ light chain‐bearing B cells, we immunized mice with low doses of lipopolysaccharide (LPS) coupled to antibodies specific for the constant region of λ1 chains. Analyses of the sera of treated mice revealed that LPS anti‐λ1 selectively triggers λ‐ but not ϰ‐bearing B cells to high rate Ig secretion. Surprisingly, immunized SJL mice (r phenotype) mounted a λ+ response comparable to that detected in mice with the phenotype. Because we could definitively establish that the majority of the induced antibodies belong to the λ1 subtype, our results have strong implications both for the study of the repertoire of λ1‐bearing B cells and for our understanding of the functional mechanism(s) by which the rλ1 locus acts.
Activation of macrophage tumor cytotoxicity by the synergism of two T cell‐derived lymphokines: Immune interferon (IFN‐γ) and macrophage cytotoxicity‐ inducing factor 2 (MCIF2)
Authors: Hamann Ute , Krammer Peter H.
Published: February 2007

This report describes the role of T cell‐derived lymphokines in induction of macrophage (Ma) tumor cytotoxicity. M tumor cytotoxicity was tested with the murine Ma‐like tumor PU5‐1.8. This MΦ‐like tumor reacts to stimulation with T cell‐derived lymphokines and shows tumor cytotoxicity comparable to resident peritoneal MΦ. The experiments demonstrate that immune interferon (IFN‐γ) secreted by T cell clones in limiting dilution microcultures was insufficient to induce MΦ tumor cytotox icity. Induction of MΦ tumor cytotoxicity by T cell‐derived supernatants with quantities of IFN‐γ undetectable in the IFN assay, however, was completely inhibited by an antiserum raised against recombinant IFN‐γ. Taken together, these results could be thus explained: (a) a T cell‐derived lymphokine distinct from but serologically cross‐reactive with IFN‐γ induces MΦ tumor cytotoxicity, and (b) IFN‐γ activity in supernatants positive for induction of MΦ tumor cytotoxicity escapes detection in the IFN assay but can still be inhibited by the anti‐IFN‐γ antiserum in the MΦ tumor cytotoxicity assay. The latter explanation requires positive evidence for another T cell‐derived lymphokine inducing MΦ tumor cytotoxicity together with IFN‐γ. This lymphokine was found in the supernatant of T cells in limiting dilution cultures and a long‐term T cell clone and was called Mγ cytotoxicity‐inducing factor 2 (MCIF2). MCIF2 synergizes with IFNγ induction of MΦ tumor cytotoxicity; IFN‐γ and MCIF2 alone were ineffective.
Cytotoxic T cell clone‐specific monoclonal antibodies used to select clonotypic antigen‐specific cytotoxic T cells
Authors: Acha‐Orbea Hans , Zinkernagel Rolf M. , Hengartner Hans
Published: February 2007

Two rat monoclonal antibodies (mAb) have been produced which recognize a clonespecific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3F9. CTL clone 3 F9 of BALB/c origin is specific for H‐2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H‐2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44‐22‐l(IgG2a) and 46‐6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44‐22‐1 has been used to sort cells from a primary MLC BALB/c anti‐ H‐2Db by fluorescence‐activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype‐specific determinants. The lymphocytes reactive with 44‐22‐1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44‐22‐1 and 46‐6 B5 whereas the sorted 3 F9 clonotype‐positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS‐sorted clonotype‐positive culture show all the same properties and are identical with clone 3F9 with respect to antigen‐specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.
T cell‐replacing factor in specific antibody responses to influenza virus by human blood B cells
Authors: Cailard Robin E. , Booth Roger J. , Brown Marion H. , McCaughan Geoffrey W.
Published: February 2007

In man, B cell maturation factors obtained from T cells or T cell lines have been shown to induce antibody formation in mitogen or anti‐immunoglobulin activated B cells, and in some continuous B cell lines, but the relationships between these factors and B cell differentiation factors in antigen‐specific antibody responses is unclear. We have now shown that supernatants from phytohemagglutinin‐activated tonsil cells, or from the Gibbon Ape T cell line ML A‐144, can substitute for T cells in the specific antibody response by human blood B cells to influenza virus. Thus, T cell‐depleted non‐rosette‐forming (E−) cells prepared from peripheral blood mononuclear cells made antibody when cultured with antigen and factor together, whereas control cultures of E− cells with either antigen or factor alone did not. Moreover, E− cells cultured with factor and influenza virus strain A/X31 made antibody to A/X31, but not the non‐cross‐reacting strain, B/HK (and vice versa)showing that the response was antigen specific. The activity in these supernatants, therefore, fulfilled the functional definition of T cell‐replacing factor (TRF). The possibility that interleukin 2 (IL2) present in the TRF‐containing supernatants was expanding residual T cells in the E− preparations to provide normal T cell help was excluded in three different ways. First, E− cells depleted of T (Leu4+) cells to undetectable levels made normal amounts of antibody when cultured with antigen and TRF. Secondly, a limiting dilution technique was employed to show that help in cultures of E− cells and TRF was not mediated by antigen‐specific T helper cells. Thirdly, TRF‐containing supernatants depleted of IL2 retained activity, whereas purified IL2 was inactive. Preliminary purification of TRF by gel filtration on Ultrogel AcA54 columns showed that all the activity eluted in a single peak between 35000 and 43000 molecular weight. This distinguishes human TRF from IL2 and from other B cell maturation factors with a molecular weight range of 15000–20000 which act on continuous B cell lines.In addition to TRF, supernatants from phytohemagglutinin‐activated tonsils also contained a factor which could induce polyspecific IgM production, but only in cultures containing significant numbers of T cells. This additional activity may have been due to IL2, and provides an explanation for the apparent T cell‐dependent effects sometimes observed in experiments designed to test B cell differentiation factors on T cell‐ depleted normal B cells.
Rat natural killer cell and cytotoxic T cell lysis of H‐2‐negative murine embryonal carcinoma cells
Authors: Bell Susan M. , Stern Peter L.
Published: February 2007

H‐2‐lacking murine embryonal carcinoma (EC) cells have been proposed as universal targets for natural killer (NK) effectors from different species because their killing appeared to be uncomplicated by potential T cell effector mechanisms (Stern, P. L. et al., Int. J. Cancer1981. 27: 679). While some previous studies had shown that murine cytotoxic T cells were unable to lyse EC cells, rat T killers are shown here to be active against these targets and to be distinguishable from NK cells.

Percoll density fractionation of rat peripheral blood lymphocytes enriches in parallel for NK‐mediated lysis of both EC or YAC target cells. These NK cells, unlike T cells, do not mediate lectin‐dependent and cell‐mediated cytotoxicity (LDCC) of NK‐insen‐ sitive target cells. This procedure is thought to reveal the total cytolytic potential of stimulated T cell populations, regardless of specificity. In contrast to previous results with mice, we found that allogeneically primed rat cytotoxic T cells can kill murine EC cells in LDCC and, further, that rat cytotoxic T cells, generated by stimulation with mouse spleen cells in vitro, can lyse murine EC cells directly.

This demonstration of T cell lysis of EC cells suggests that either there is a novel mechanism of lysis operating without requirement for major histocompatibility com‐ plex (MHC) structures, or EC cells express some hitherto unidentified MHC‐like structures on their cell surface.
Clones of B lymphocytes in individual follicles of the bursa of Fabricius
Authors: Pink J. Richard , Vainio Olli , Rünbeek Anne‐Marie
Published: February 2007

To discover whether individual bursal follicles can contain clones of B lymphocytes, we estimated the numbers of lymphoid cell precursors populating single follicles in two types of chicken chimera. The first type was produced by establishing parabiotic connections between blood vessels of embryo chorioallantoic membranes. Under these conditions, and most likely during normal development, most follicles are populated by more than one, but less than ten, precursor cells. However, in a second type of chimera, a cyclophosphamide‐treated chick reconstituted with normal bursal cells, most follicles in the reconstituted bursa are clonal (their lymphocytes are derived from a single precursor cell). Individual follicles can readily be isolated from bursae of reconstituted birds and should be useful in studies of B cell development.
Capacity of small B cell‐enriched populations to stimulate mixed lymphocyte reactions: marked differences between irradiated vs. mitomycin C‐treated stimulators
Authors: Webb Susan R. , Li Jane Hu , Wilson Darcy B. , Sprent Jonathan
Published: February 2007

To investigate whether small B cells can stimulate mixed lymphocyte reactions (MLR), highly purified populations of large vs. small B cell fractions were tested for their capacity to evoke MLR across Mls vs. H‐2 barriers. Large B cell fractions stimulated high MLR to Mls and H‐2 determinants, irrespective of whether the stimulators were exposed to irradiation or pretreated with mitomycin C. In accord with the findings of others, irradiated small B cell fractions proved to be very poor stimulators of MLR. Significantly, however, mitomycin C‐treated small B cell fractions elicited high MLR, particularly to Mls determinants.

The finding that small B cell fractions treated with irradiation are poor stimulators of T cells correlates with the known radiosensitivity of B cells. In this respect, the widely held view that small B cells do not have antigen‐presenting cell (APC) function rests largely on studies with irradiated B cells. The present finding that T cells respond well to small B cells treated with mitomycin C, however, indicates that small B cell fractions do have APC function. Whether the APC function of small B cells reflects a response to resting B cells per se rather than to cells undergoing activation in vitro, however, remains to be ascertained.
Lectin separation of nonlymphoid suppressor cells induced by total lymphoid irradiation
Authors: Morecki Shoshana , Weigensberg Marilyn , Slavin Shimon
Published: February 2007

Suppression of the mixed lymphocyte reaction (MLR) exerted by splenocytes derived from mice treated with fractionated total lymphoid irradiation (TLI, 200 rds × 8) was analyzed by various criteria in order to characterize the phenotype of the cell type(s) responsible for suppression. TLI‐induced suppressor cells could not be eliminated by removal of cells bearing surface immunoglobulin, Thy‐1, Lyt‐2 and TL, and thus could not be ascribed to lymphocytes of the B or T cell lineage. Suppressor cells were large, and nonadherent to nylon wool, Sephadex G‐10 and plastic surfaces. Suppressor activity of TLI splenocytes was predominantly located in fractions of cells bearing receptors for soybean agglutinin (SBA), peanut agglutinin (PNA) or both lectins. SBA+, PNA+′, sequentially agglutinated (SBA followed by PNA) SBA+PNA+ and (PNA followed by SBA) PNA+SBA+ suppressor cells were radioresistant upon exposure to 1000 rds in vitro. Cells bearing the receptor for PNA but lacking that for SBA (PNA+SBA−) had sharply reduced suppressor activity. However, a radiosensitive PNA− suppressor cell subset was also documented in the spleen of TLI‐treated mice. Thus, suppressor cells could best be physically separated from nonsuppressors by the SBA lectin. SBA+ suppressor cells were found, by scatter analysis, to include the population of large cells characteristic of TLI splenocytes, whereas SBA− cells were much smaller and almost exclusively devoid of suppressive capacity. The PNA receptor was found to further dissect the SBA+ suppressor cells into two distinct subpopu‐lations: radioresistant SBA+PNA+ cells and radiosensitive SBA+PNA− cells. In summary, we suggest here the presence of at least two suppressive populations induced by TLI: (a) radioresistant SBA+, PNA+, SBA+PNA+ or PNA+SBA+ cells, and (b) radiosensitive PNA− and SBA+PNA− cells. Similar subsets of MLR suppressor cells can be isolated from normal bone marrow cells and splenocytes of nude mice, suggesting that suppression is mediated by large, immature, nonlymphoid cells which might migrate from shielded bone marrow compartments into the spleen of TLI‐treated mice.
Function of alloproliferative T lymphocyte clones correlates better with their class II recognitive specificity than with their cell surface phenotype
Authors: Pawelec Graham , Schneider E. Marion , Wernet Peter
Published: February 2007

Alloproliferative primed lymphocyte typing (PLT) clones recognizing determinants associated with HLA‐DR/Dw, SB, MB, or novel “SB‐like” gene products were screened for their ability to suppress lymphoproliferative responses in primary and secondary mixed lymphocyte cultures (MLC), and for their surface marker pheno‐types. Two nonsuppressive HLA‐D specific PLT clones were OKT4−, OKT+ whereas all others possessed an OKT4+, OKT8−, Leu‐8− phenotype. All clones secreted interleukin 2 (IL2) after specific stimulation. The eight PLT clones specific for “SB‐like” antigens strongly suppressed MLC, whereas only one of 35 DR/Dw‐specific, and none of 20 SB‐specific PLT clones did so. Suppressive activity of such PLT clones was not restricted by major histocompatibility complex products, was radioresistant (20 Gy), and was not caused by absorption of IL2 or by cytotoxicity of the cloned cells. Suppressive clones exerted their effects directly on proliferating T cells, as assessed by their ability to prevent growth of cloned PLT cells stimulated by B cell lines, and their ability to block primary MLC even when added 96 h after the start of the 144‐h culture. Culture supernatants from suppressive, but not from nonsuppressive, PLT clones also strongly and nonspecifically inhibited lymphoproliferative responses. The suppressive factor(s) was not dialyzable, not sensitive to pH 2 or heat treatment and not cytotoxic. Thus, all T cell clones proliferating against novel “SB‐like” but not SB antigens, as well as rare clones specific for D region determinants, possess powerful nonspecific suppressive activities dissociated from their “helper‐related” OKT4+, OKT8‐, Leu8−, IL2‐secreting phenotypes.
Tumor promoter phorbol esters induce unresponsiveness to antigen and expression of interleukin 2 receptor on T cells
Authors: Ando Istvan , Hariri Gita , Wallace Diana , Beverley Peter
Published: February 2007

The tumor promoter 12‐O‐tetradecanoylphorbol l3‐acetate induces the expression of the interleukin 2 receptor and the disappearance of the T3 complex in a T cell hyb‐ridoma, T cell clones, thymic and peripheral T cells and T cell tumors. These changes are accompanied by an increased sensitivity to interleukin 2 and antigen‐specific unresponsiveness in T cell clones.
Inhibition of hapten‐specific cytotoxic T cell recognition by monoclonal anti‐hapten antibodies
Authors: Pircher Hanspeter , Zinkernagel Rolf M. , Hengartner Hans
Published: February 2007

The T cell‐mediated Cytotoxic response against autologous cells modified with the sulfhydryl reagent I‐AED (N‐iodoacetyl‐N′‐(5‐sulfonic‐l‐naphthyl) ethylene diamine) is hapten specific and H‐2 restricted (Levy, R. B., Shearer, G. M., Richardson, J. C. and Henkart, P. A., J. Immunol. 1981. 127: 523). We have produced a monoclonal antibody (V‐6‐3, IgM) which binds to AED‐modified cells and proteins. Competition experiments by free hapten indicated that the binding was AED specific. The effect of the mAb on AED‐specific cytotoxic T cell recognition at the effector and induction stage has been examined. Anti‐AED mAb inhibited the cell‐mediated lysis of some but not all AED‐specific, H‐2b‐restricted long‐term cytotoxic T cell clones and of bulk‐cultured C57BL/6 anti‐AED‐self effector cells. This blocking was not due to nonspecific agglutination of targets since lysis of AED‐modified target cells by alloreactive effector cells was not affected by this mAb under comparable conditions. Furthermore anti‐AED mAb specifically inhibited the antigen‐induced proliferation of AED‐specific long‐term cytotoxic T cell clones and the generation of AED‐specific cytotoxic effector cells in secondary cultures. This monoclonal anti‐AED antibody bound to cells modified by the recently described aminoreactive reagent AED‐NH2 (Takai, Y., Mizuochi, H., Fujiwava, H. and Hamaoka, T., J. Immunol. 1984. 132: 57); these same target cells were, however, not lysed by AED‐SH‐specific cytotoxic T cell clones.
Isotypically restricted activation of B lymphocytes by lactic dehydrogenase virus
Authors: Coutelier Jean‐Paul , Van Snick Jacques
Published: February 2007

Lactic dehydrogenase elevating virus (LDV) was found to selectively stimulate IgG2a synthesis in infected mice. Within one week after infection, the production of IgG2a increased nearly 50‐fold whereas that of IgM, IgA, IgGl and IgG3 remained virtually unchanged. IgG2b synthesis was also enhanced but to a lesser extent. Several observations suggested that this stimulation of IgG2 production resulted from a polyclonal B cell activation: (a) the isoelectric focusing patterns of IgG2a, before and after LDV infection were exactly the same, (b) the frequency of clones with anti‐LDV activity in hybridoma collections derived from infected mice was extremely low (less than 4/ 1000) and (c) the proliferative response elicited by LDV in unsensitized animals was comparable with that induced by lipopolysaccharide. The effect of LDV on immunoglobulin synthesis was drastically reduced in nude mice but was not affected by the X‐linked B lymphocyte defect of animals carrying the xid mutation.
Antigen‐specific suppression of anti‐influenza antibody production in man. Possible role of a membrane‐antigen complex
Authors: McCaughan Geoffrey W. , Brown Marion H. , Basten Antons
Published: February 2007

E rosette‐forming (E′) cells from human secondary lymphoid tissue were incubated with high dose influenza A virus (Mem‐Bel) in an attempt to generate antigen‐specific suppressor T cells. Suppression was assayed by transferring the antigen‐pulsed E+ cells into effector cultures consisting of E+ and E− cells stimulated with immunogenic amounts of either the inducing virus (Mem‐Bel) or the non‐cross‐reacting influenza B virus (B/HK). The transfer resulted in marked inhibition of IgG, IgA and IgM antibody production to Mem‐Be1 but not to the control antigen, B/HK virus. The suppres‐ sive effect was specific at the level of induction as well as expression since E+ cells exposed to high dose Mem‐Be1 could provide help to an effector culture containing E− cells and optimal dose of B/HK virus. However, metabolically active cells did not appear to be required for suppression. Thus, it could be elicited (a) after only 15 min incubation of E+ cells with high‐dose virus and (b) by E+ cells exposed to irradiation, incubated in the presence of metabolic inhibitors, or disrupted by repeated freeze thawing. In contrast, treatment of E+ cells with pronase reversed the suppressive effect. Interestingly, virus heated to 70 oC failed to induce suppression, while retailing the ability to elicit a normal helper response.

Suppression induced by exposure to standard amounts of high‐dose antigen was medi‐ ated by T cells of both helperiinducer (Leu‐3a+) and suppressoricytotoxic subsets (Leu‐2a+), but not by B cells. Two groups of observations pointed to the B cell as the target of suppression. First, suppression could still be transferred to effector cultures in which helper T cells had been replaced by T cell‐replacing factor or suppressor T cells removed by irradiation. Second, significant inhibition of antibody production was obtained when the transfer of antigen‐pulsed E+ cells was delayed for up to 120 h after initiation of the effector culture. Taken together the results suggest that suppression in this system is due to the formation of an antigen bridge between specific receptor sites on the T cell membrane and the target. Although not dependent on triggering of metabolically active suppressor T cells the phenomenon highlights the need for care in interpreting the mechanism of suppression by high‐dose antigen and could, in addition, represent a biologically important control mechanism capable of rapid inhibition of effector T cells and B cells in sites of high antigen concentration.
Phorbol ester‐induced expression and function of the interleukin 2 receptor in human B lymphocytes
Authors: Ando Istvan , Crawford Dorothy H. , Kissonerghis Maija A. , Owen Michael J. , Beverley Peter C. L.
Published: February 2007

The expression of the interleukin 2 receptor (Tac) on normal B cells and Epstein‐Barr virus‐transformed lymphoblastoid B cell lines is induced by the tumor promoter 12‐O‐ tetradecanoyl‐phorbol‐13‐acetate (TPA). The receptor on B cells is indistinguishable from that on T cells by serological and biochemical analysis. It is functionally active so that TPA‐induced B cells respond to interleukin 2 by increased DNA synthesis. These results suggest that the expression of Tac is not exclusively restricted to T cells and that interleukin 2 may play a role in B cell differentiation.
Alloreactive cytotoxic T cells. I. Alloreactive and allorestricted cytotoxic T cells
Authors: Reimann Jorg , Heeg Klaus , Miller Richard G. , Wagner Hermann
Published: February 2007

Nylon wool‐nonadherent spleen cells from three inbred mouse strains of H‐2k (CBA), H‐2d (BALB/c) and H‐2b (C57BL/6) haplotype were co‐cultured with 2,4,6‐ trinitro‐ phenyl (TNP)‐ modified or nonmodified allogeneic stimulator cells in a limiting dilution system. Using a recently described restimulation protocol, a surprisingly large number of splenic cytotoxic lymphocyte precursors (CLP) was clonally expanded in this primary in vitro response to allo ‐ H‐2 plus TNP determinants; measured CLP frequencies ranged from 1/30 to 1/300. The lytic specificity patterns of individual microcultures (selected for a high probability of clonality) were defined by split well analysis, and were furthermore followed up in time by sequentially reassaying microcultures at different time points of in vitro incubation. This analysis revealed the following: (a) a large fraction of cytotoxic T lymphocyte clones lysed TNP‐modified but not nonmodified all ogeneic concanavalin A blast targets, i.e., were alloiestricted; (b) this was found in all 6 allogeneic strain combinations set up with b, k and d haplotype mice; (c) allorestricted lytic patterns predominated in microcultures with low numbers of responder cells per well, and at late time points of in vitro culture; (d) allorestricted lytic cultures were specific for the stimulating allogeneic H‐2 plus T NP determinant(s); and (e) allorestricted lytic patterns were also found in microcultures stimulated by nonmodified allogeneic cells. To our knowledge, these are the highest CLP frequencies yet reported in limiting dilution systems that used a specific (re)stimulation protocol and measured the lytic responses obtained in a specificity‐ controlled readout.
Interferon gamma produced by mitogen‐activated T lymphocytes does not directly mediate lymphoproliferation
Authors: Wilkinson Miles , Morris Alan , Leung Helen , Crane Isabel , Meager Anthony
Published: February 2007

Human interferon gamma (IFN‐γ) endogenously produced during mitogenic stimulation of human peripheral blood mononuclear cells or human T lymphocyte‐enriched cultures was neutralized in situ by the addition of a polyclonal antiserum (anti‐L) raised in a rabbit against partially purified natural IFN‐γ derived from peripheral blood mononuclear cells. Small decreases in mitogen‐induced [3H]thymidine incorporation (lymphoproliferation) were demonstrated under these conditions. However, an antiserum (anti‐G) raised in a sheep against highly purified recombinant IFN‐γ (E. coli‐derived) which strongly neutralized the antiviral effect of IFN‐γ either had little effect on mitogen‐induced lymphoproliferation or caused slight enhancement of mitogenesis. The interleukin 2 responsiveness of activated T lymphocytes following mitogenic stimulation was not found to be different in the presence of anti‐G to that of control cultures incubated in the presence of normal sheep serum. These results suggest that IFN‐γ is not a direct requirement for lymphoproliferation.