bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirements for growth of immature thymocytes from fetal and adult mice in vitro
Authors: Palacios Ronald , Boehmer Harald Von
Published: February 2007

We report here defined culture conditions that allow reproducibly the growth of the majority of immature thymocytes from both fetal (14–15 days of gestation) and adult mice. The combination of phorbol myristate acetate (PMA), ionomycin and recombinant interleukin 2 (IL2) is both sufficient and necessary to induce growth of about 1/6.2 (range 1/3–1/9) and 1/4.3 (range 1/2–1/7) immature thymocytes from adult and fetal mice, respectively, in serum‐free cultures. Several other combinations tested (e.g. PMA + IL2, concanavalin A + IL2) were poorly or not active. None of the agents tested alone (PMA, ionomycin, concanavalin A, pokeweed mitogen, IL2) had any effect. We found no evidence for a role of IL1 and IL3 on growth of these cells. The growth of activated immature thymocytes from either fetal or adult mice was inhibited by a monoclonal antibody against mouse IL2 receptors.

Under the same conditions that stimulated growth of most immature thymocytes, they did not mature into cells expressing Lyt‐2, L3T4 or T cell antigen receptor (KJ16) after 7 to 15 days of continuous proliferation in culture. Nor did they give rise to cells with cytolytic activity after 7–9 days of culture. In some but not all experiments cultures of immature thymocytes from adult mice but not from fetal mice generated cells (1 out of 120–310) with helper function for B lymphocytes.

While we confirmed here that ∼50–70% freshly isolated immature thymocytes express receptors for IL2, our results indicate that these cells need to be activated (by e.g. PMA + ionomycin) to respond to IL2. A possible mechanism to account for the expression of nonfunctionally competent IL 2 receptors is proposed and our results concerning the maturation of immature thymocytes in vitro are discussed.
High antigen concentration inhibits T cell proliferation but not interleukin 2 production: Examination of limiting dilution microcultures and T cell clones
Authors: Ceredig Rhodri , Corradin Giampietro
Published: February 2007

The effect of high antigen dose on the activation of cytochrome c peptide‐primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide‐specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 moduction by these clones was not impaired regardless of the antigen concentrations used.
Monocyte‐dependent induction of proliferation of human peripheral T cells by recombinant interleukin 2
Authors: Roosnek Eddy E. , Brouwer Mieke C. , Kipp J. Bart , Aarden Lucien A.
Published: February 2007

The in vitro proliferation of human peripheral lymphocytes induced by interleukin 2 (IL 2), a product of cDNA, cloned in E. coli has been studied. The maximal mitogenic signal is given by concentrations ≦2.5 U/ml. Due to growth factor consumption, at least 10 U/ml are needed to maintain a logarithmic response until day 6. The anti‐Tac antibody, directed against the IL2 receptor, effectively blocks this response, but we could not obtain a decrease of IL2‐reactive cells by depletion of putative in vivo activated Tac+ cells, using this antibody and a fluorescence‐activated cell sorter.

Depletion of Leu 7+ and Leu 11b+ cells does not cause a decrease of the response, which indicates that the responding cells are not confined to the natural killer lineage. By simultaneous staining of cell‐surface markers and DNA, the nature of the proliferating cell was determined. More than 90% of the dividing cells expressed HLA class II and the Tac antigen, whereas the lymphocyte populations, defined by the surface markers Leu 2, Leu 3, Leu 4, Leu 7 and Leu 11b, were all represented in the dividing cells. The magnitude of the response was proportional to the number of monocytes present in the culture. Depletion of monocytes completely abrogated the response, whereas an increase in the number of monocytes to a 1:1 ratio with lymphocytes caused a 2‐fold increase in proliferation. However, purified T cells do proliferate to IL 2 when cultured in the presence of a supernatant that was harvested from a 2‐day culture of adherent monocytes. The proliferation‐inducing activity in the supernatant eluted with apparent molecular weights of 15 000 and 75 000 on an Ultrogel AcA‐54 column. Therefore, we conclude that in vitro, in the presence of an IL 1‐like activity produced by monocytes, IL2 is mitogenic for a population of T cells.
Autoimmunity to Thy‐1
Authors: Thomas D. Brian , Giguere Vincent , Graham Christine M. , Oliveira Odete L. P.
Published: February 2007

Conditions are described for the induction of autoimmunity to Thy‐1 and a large panel of monoclonal CBA and AKR autoantibodies has been characterized. These reveal a hitherto unrecognized complexity at the Thy‐1 locus and evidence for intragenic control. Epitopes recognized by the autoantibodies differed in species and tissue distribution from alloantigenic determinants but their specificity was confirmed by transfection studies with the cloned Thy‐1b gene. The quantitative and qualitative differences in epitope expression between Thy‐1a and Thy‐1b alleles were inherited as single Mendelian traits and in the absence of recombinants, suggesting translational control.
Immune response to diphtheria toxin and to different CNBr fragments: Evidence for different B and T cell reactivities
Authors: Triebel Frédéric , Autran Brigitte , De Roquefeuil Sabine , Falmagne Paul , Debré Patrice
Published: February 2007

The antigenic structure of the diphtheria toxin has been studied in man. B and T cell responses to diphtheria toxoid and to different fragments of the toxin molecule were analyzed in 4 individuals one month after booster immunization. Studies on the B cell response showed that: (a) part of the response was directed against assembled topographic sites; (b) 80% of the response was directed against determinants present on fragment A; (c) the few determinants present on the CNBr peptides of B cross‐react with determinants present on A; and (d) reduction of the second disulfide bridge of fragment B diminishes the response. In contrast to the antibody response, most of the T cell reactivity was directed against the B fragment or CNBr peptides from this fragment. Analysis of the fine specificity of T lymphocyte clones revealed that some CNBr fragments share common T cell determinants. These studies indicate that T and B cell determinants are differently distributed on the molecule and that large cross‐reactivities that are not explained by the analysis of the amino acid sequence could be found at the B and T cell level.
Lymphocyte membrane lipid composition and mitogen responsiveness in chickens: Role of membrane “fluidity”
Authors: Traill Karine N. , Ratheiser Klaus , Pfeilschifter Ruth , Wick Georg
Published: February 2007

After establishing optimal conditions for measuring the membrane lipid packing density (“fluidity”) of chicken peripheral blood lymphocytes, the fluidity was modulated in vitro by incubation in cholesterol or phospholipid (“active lipid”, AL)‐enriched serum‐free tissue culture medium. The effect of these lipids on mitogen responsiveness was then investigated, the aim being to determine whether the observed enhancement/suppression was membrane mediated, i.e. explainable by fluidity changes.

Chicken peripheral blood lymphocytes exhibited no requirement for exogenous cholesterol; low concentrations did not affect the mitogen response while the higher concentrations, which induced a measurable decrease in membrane fluidity, were usually mildly suppressive. Pre‐incubation did not increase this suppressive effect and we believe it not to be membrane mediated.

AL, at low concentrations which induced no changes in membrane fluidity, prolonged the phytohemagglutinin response, enhancement being evident only after the peak; we interpret this as a nutrient effect. At the higher concentrations, which induced large increases in fluidity, a transient enhancement was followed by suppression; suppression was delayed in onset when AL was added 4 h after phytohemagglutinin stimulation. It is therefore an early event which may be mediated through changes in membrane fluidity.
T cells bearing unique membrane antigen and their regulatory role in humoral immune response in mice
Authors: Ishida Yasuo , Sekigawa Iwao , Hamaoki Masaru , Takenouchi Toshinao , Yumura Wako , Sato Hidetoshi , Shirai Toshikazu
Published: February 2007

The mutant lpr gene causes a generalized massive T cell proliferation and an associated autoimmune disease in mice. The lpr T cells bear unusual membrane phenotypes and play a causative role in the pathogenesis of autoimmune disease through excess production of some lymphokines. Therefore, the “aberrant” T cells in lpr mice may be normal regulatory T cells in non‐lpr‐bearing mice, if nonproliferative. We thus attempted to produce a xenogeneic monoclonal antibody (ALP‐1) directed against the lpr T cells. The ALP‐1 monoclonal antibody reacted with the antigen Lp‐1 distributing on about one half of the proliferating lymph node cells from MRL/Mp‐lpr/lpr mice but not on the cells from non‐lpr‐bearing mice. Nevertheless, a significant number of Lp‐1+ cells appeared in the lymph node and spleen cells from these normal mice after in vitro stimulation with mitogens. Functional studies revealed that either the treatment with ALP‐1 plus complement of the antigen‐primed splenic T cells from non‐lpr‐bearing mice or the addition of ALP‐1 alone all but completely abolished the development of plaque‐forming cells against sheep red blood cells in T‐B cell co‐culture. Pretreatment of spleen cells with ALP‐1 plus complement, however, did not affect other in vitro T cell functions such as mitogen‐induced proliferative responses, interleukin 2 production, response in mixed lymphocyte culture (MLC) and the MLC‐induced cytotoxic T cell activities. Thus, Lp‐1+ T cells in normal mice are unique T cells that may play an important regulatory role in humoral immune response.
Self‐tolerance to host and donor following HLA‐mismatched bone marrow transplantation
Authors: De Villartay Jean‐Pierre , Griscelli Claude , Fischer Alain
Published: February 2007

The transplantation of T cell‐depleted HLA‐haploidentical bone marrow can correct the severe combined immunodeficiency disease (SCID) caused by the inherited absence of T lymphocytes. Despite a different environment, no severe graft‐vs.‐host reaction occurred and engrafted T lymphocytes became functional. We have studied tolerance of engrafted T lymphocytes to donor and host HLA antigens in four SCID patients who have been transplanted with bone marrow from one of their HLA‐haploidentical parents. Graft‐vs.‐host reaction was prevented by T cell depletion of infused bone marrow using E rosetting and by in vivo administration of cyclosporine A. Subsequent to bone marrow transplantation (BMT), the engrafted T lymphocytes were shown to be unresponsive in vitro towards host cells collected prior to BMT. Generally, this tolerance could not be explained by a suppressive mechanism. Nevertheless, in one patient suppressive cells were found transiently. In contrast to the early appearance of a tolerance towards host, a reactivity of engrafted donor cells towards donor was always observed within the first 300 days post‐grafting. This auto‐reactivity was mediated by T cells of donor origin and its targets were HLA class II molecules (at least HLA‐DR and DQ). The progressive disappearance of this auto‐reactivity was correlated with the engraftment of Ia‐positive cells (monocytes plus B lymphocytes) of donor origin and the achievement of complete immunological reconstitution. In the patient showing the strongest autoreactivity, a donor‐specific T cell line has been grown which was shown to specifically inhibit the proliferative response of donor lymphocytes. Concomittantly, the immunological reconstitution remains poor in this patient. These data suggest that tolerance to HLA class II molecules is dependent on the presence of the relevant HLA class II molecule‐expressing cells allowing the elimination or the suppression of T lymphocytes specifically directed at these molecules.
Limiting dilution analysis of the B cell compartment in human bone marrow
Authors: Hibi Toshifumi , Dosch Hans‐Michael
Published: February 2007

Mononuclear cells (MNC) obtained from adult rib specimen were investigated for their capacity to produce immunoglobulin (Ig) in vitro. Using limiting dilution analysis 3 populations of B lineage cells could be distinguished. The first produces Ig in culture without any intentional activation. These cells are strictly nonproliferating and sustain extraordinary secretory rates of 5 × 107‐10 × 107 molecules IgG, IgA or IgM/cell/h for at least 2 weaks. They occur at frequencies of 1 × 10‐4‐10 × 10−4 (marrow MNC) or represent 1–5% of marrow B cells. Following exposure to infectious Epstein‐Barr virus (EBV), these cells do not (a) proliferate, (b) express EBV‐determined nuclear antigens (EBNA), (c) enhance their secretory rates or (d) show secretory activity for prolonged time. These cells are therefore EBV resistant. If these cells produced their antibody at similar rates in vivo, then their frequencies would suggest that they could provide up to 2/3 of daily synthetic rates for IgG and IgA and 20–30% of the daily IgM production. The second population of marrow B cells is exclusively committed to IgM production. Following exposure to EBV these cells proliferate, express EBNA and begin to secrete IgM. The third population represents 90% of marrow B cells. In our hands, these cells are unable to produce Ig in vitro.
Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein‐Barr virus‐immortalized B cells
Authors: Polke Christine R. , Lowenthal John W. , Roth Stephan A. , Rohwer Peter , MacDonald H. Robson , Kalden Jochen R.
Published: February 2007

The enhanced expression of interleukin 2 (IL2) receptors by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) on sublines of an Epstein‐Barr virus‐immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti‐Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2‐binding studies revealed that TPA induced an increase in not only the number of IL 2 receptors per cell but also the affinity of the receptors for IL 2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells.

Furthermore, TPA‐induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme‐linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed.
Only Tα or Tγ cells can be triggered by IgG or IgA to suppress the production of the matching Ig class
Authors: Bich‐Thuy LĚ Thi , Revillard Jean‐Pierre
Published: February 2007

A short exposure of human peripheral blood mononuclear cells (PBMC) to heat‐aggregated IgG (or IgA) was found to inhibit pokeweed mitogen (PWM)‐induced human B cell differentiation into IgG (or IgA)‐producing cells. The present study was designed to investigate the nature of the cells inducible by aggregated (Agg)‐IgG or Agg‐IgA into isotype‐specific suppressors. When T cell‐enriched suspensions were exposed to Agg‐IgG or Agg‐IgA, then thoroughly washed, and cultured with autologous nonrosetting cells in the presence of PWM, a selective suppression of the generation of cells synthesizing and secreting IgG or IgA, respectively, was observed. Conversely, no inhibition was induced by T‐depleted PBMC treated in exactly the same way. Unlike Agg‐IgG, Agg‐F(ab′)2 fragments did not trigger suppression. Finally, in vitro X‐irradiation of T cells pretreated with Agg‐IgG or Agg‐IgA abrogated their suppressive activity. It is concluded that among cells bearing Fcγ or Fcα receptors, only T cells were inducible into suppressor cells by the binding of the matching immunoglobulin class.
Viral recognition by influenza A virus cross‐reactive cytotoxic T (Tc) cells: The proportion of Tc cells that recognize nucleoprotein varies between individual mice
Authors: Pala Pietro , Townsend Alain R. M. , Askonas Brigitte A.
Published: February 2007

L cells (H‐2k) transfected with DNA coding for the A/NT/60/68 influenza nucleoprotein (NP) gene have been found previously to provide a target cell for cytotoxic T (Tc) cells. In this report we have studied the repertoire of cytotoxic memory cells in mice of different strains primed by infection with several influenza A subtypes. L cells transfected with NP and Db genes enabled us to estimate the proportion of A virus cross‐reactive Tc cells specific for NP.

The most striking feature of our results was variation in the frequency of NP‐specific Tc cells between individuals of an inbred mouse strain. Occasional individuals, although showing a strong A virus cross‐reactive Tc response, had no A virus cross‐reactive Tc cells that recognized NP. The NP‐directed Tc repertoire represented between 0‐40% of lysis of target cells infected with a heterologous type A virus. Thus, a significant proportion of A virus cross‐reactive Tc cells must have different specificities for type A influenza virus. In C3HH‐2o2 mice, Dk is a low responder allele for NP recognition by Tc cells.
The cellular basis for the induction of antigen‐specific T8 suppressor cells
Authors: Morimoto Chikao , Letvin Norman L. , Distaso Judy A. , Brown Howard M. , Schlossman Stuart F.
Published: February 2007

The cellular basis for the generation of antigen‐specific T8 suppressor cells with high doses of antigen has been studied. We separated the T4 subset of human T cells into T4+2H4+ and T4+2H4− subpopulations with a recently developed monoclonal anti‐2H4 antibody. T8 cells could be consistently activated to suppress a primary anti‐2,4‐dinitrophenyl (DNP) antibody response in vitro with unfractionated T4 cells or with the T4+2H4+ subset but not the T4+2H4− subset. In contrast, the T4+2H4− subset functioned as the helper inducer for the anti‐DNP antibody response. With keyhole limpet hemocyanin (KLH)‐stimulated T4+2H4+ cells we could efficiently induce antigen‐specific suppressor activity of fresh T8 cells. In contrast, the T4+2H4+ subset could not effect suppression in the absence of T8 cells. Our findings indicate that the T4+2H4+ subset of human T cells is the suppressor inducer of specific T8 cells in an antigen‐specific DNP‐KLH system.
Antigen‐specific suppression of human antibody responses by allogeneic T cells II. Cell interactions involved in the generation of suppression
Authors: Callard Robin E. , Tiernan Shane L. , Winger Carrie M.
Published: February 2007

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBMC). Antibody production in these cultures was profoundly suppressed by the addition of allogeneic T cells with the surface phenotype Leu2a+ (CD8+), Leu8−. Suppression by allogeneic T suppressor (Ts) cells required interactions only between T‐depleted B (E−) cells and allogeneic Leu2a+. No evidence was obtained for T‐T cell interactions, or for Ts inducer cells similar to those described for nonspecific antibody responses to pokeweed mitogen. Moreover, allogeneic E+, or allogeneic Leu2a+ cells were able to suppress specific antibody responses by E− cells when help was provided by T cell‐replacing factor showing that the target of suppression was the responding E− cells, and not T helper cells. In contrast to allogeneic T cells, allogeneic E− cells did not suppress antibody production when added to cultures of unfractionated PBMC (E− + E+). That is, Ts cells activated to allogeneic E− were unable to suppress antibody production by the syngeneic E− cells present in the same culture tube. This result shows that alloactivated Ts cells were specific for the allogeneic E− target cells, and that suppression was not mediated by nonspecific allogeneic effects. Allogeneic Ts cells therefore differ from Ts cells in pokeweed mitogen responses by their specificity, and by their activation in the absence of Ts inducer cells.
Kinetics of cellular oncogene expression in mouse lymphocytes: I. Expression of c‐myc and c‐rasHa in T lymphocytes induced by various mitogens
Authors: Schneider‐Schaulies Jurgen , Hünig Thomas , Schimpl Anneliese , Wecker Eberhard
Published: February 2007

Murine splenic T lymphocytes display maximal cellular myc gene (c‐myc) expression already 3 h after concanavalin A stimulation and subsequent down‐regulation before the onset of DNA synthesis. Stimulation by leucoagglutinin in the presence or absence of interleukin 2 leads to only low initial levels of c‐myc‐specific RNA which, however, increase later on. A similar pattern of c‐myc expression is shown by the Lyt‐2+ T cell subpopulation stimulated with either concanavalin A or leucoagglutinin in the presence of interleukin 2. Although [3H]thymidine incorporation was identical, the leucoagglutinin‐stimulated Lyt‐2+ T cells were void of any demonstrable c‐myc‐specific RNA at 3 h post‐stimulation. Thus, the kinetics of c‐myc expression in mouse T lymphocytes are not at all uniform, but depend on the mitogen and the subpopulation. In contrast, levels of c‐rasHa‐specific RNA were always low at early times, always increased towards the onset of DNA synthesis and down‐regulation was not observed.
Induction of murine T cell lymphoma expressing specific cytotoxic activity
Authors: Schaaf‐Lafontaine Nicole , Martin Marie Thérèe , Boniver Jacques
Published: February 2007

The present work reports the establishment of an antigen‐specific cytotoxic T cell lymphoma line after immortalization with a murine leukemia virus. Lymph node cells from mice bearing a transplanted syngeneic MCA sarcoma were infected in vitro with radiation leukemia virus and injected intrathymically into congeneic recipient mice. Some lymphomas of donor origin were established as permanent continuous cell lines in vitro. One of them, NS8, expressed Thy‐1.2, Lyt‐1, Lyt‐2 and peanut agglutinin surface markers. These cells were cytotoxic in vitro for the tumor cell line corresponding to the immunizing MCA sarcoma. No significant cytolytic activities against other syngeneic or allogeneic sarcomas or lymphomas, nor against the mastocytoma P815 were observed. After several months of in vitro propagation, the specificity of the cytotoxic activity had degraded and the level of Lyt‐2 or peanut agglutinin receptors dropped. These characteristics were restored with a single in vivo passage. Thus, murine leukemia virus can be used to immortalize antigen‐specific cytotoxic T cells.
Macrophage activation by trehalose dimycolate Requirement for an expression signal in vitro for antitumoral activity; biochemical markers distinguishing primed and fully activated macrophages
Authors: Grand‐Perret Thierry , Lepoivre Michel , Petit Jean‐François , Lemaire Geneviève
Published: February 2007

It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacteriurn tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin‐free culture conditions. TDM‐elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1‐10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate‐elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 μg/ml), MDP (0.2‐1 μg/ml) and the ionophore A23187 (5 μM) can deliver the activation signal to TDM‐primed macrophages.

Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and β‐galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2.

However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D‐[2‐3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation‐associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity.
Human natural killer cell lysis of virus‐infected cells. Relationship to expression of the transferrin receptor
Authors: Borysiewicz Leszek K. , Graham Shek , Sissons J. G. Patrick
Published: February 2007

Natural killer (NK) cells lyse tumor and virus‐infected cells yet the nature of the target structure they recognize is unknown. A normal host cell glycoprotein, the transferrin receptor (TfR), has been proposed as a target structure on tumor cells. We therefore investigated whether changes in the number or physiological recycling of the TfR, consequent on virus infection, were related to the differential susceptibility of virus‐infected cells to NK lysis. There was a direct correlation between TfR expression, susceptibility to NK lysis and ability to act as cold target competitors, for human fibroblasts infected with RNA and DNA viruses (cytomegalovirus, herpes simplex, polio, vaccinia and Semliki Forest virus). The NK lysis of human cytomegalovirus‐infected fibroblasts was studied in more detail. NK lysis was increased coincident with human cytomegalovirus early antigen expression and this susceptibility to lysis was associated with increased total and recycling TfR but only a slight increase in surface TfR expression. In addition, susceptibility of uninfected human fibroblasts to NK lysis directly correlated with TfR number. However, we were unable to inhibit NK lysis by either excess iron‐saturated Tf or affinity‐purified TfR. We conclude that there is a direct correlation between total TfR expression and susceptibility to NK lysis of human virus‐infected cells; however, the NK target structure on virus‐infected cells is probably not the TfR itself.
Antigen presentation is a function of all B cell subpopulations separated on the basis of size
Authors: Jelachich Mary Lou , Lakey Ellen K. , Casten Lisa , Pierce Susan K.
Published: February 2007

Purified splenic B cells from nonimmune mice were separated by counterflow centrifugal elutriation into 6 subpopulations containing cells of discrete sizes ranging from 119 to 200 μm3. B cells of each subpopulation were competent to process and present a native globular protein antigen, cytochrome c, to a cytochrome c‐specific T cell hybrid. In all cases, the B cells' antigen‐presenting function was radiation sensitive and did not require T cells or T cell products, since B cells fixed with paraformaldehyde effectively presented a carboxyl‐terminal peptide fragment of cytochrome c containing the T cell determinant. Furthermore, the antigen‐presenting function of B cells of each subpopulation was augmented by treatment with submitogenic doses of the F(ab')2 fragment of rabbit anti‐mouse Ig antibodies, in that 10‐30‐fold fewer B cells were required and higher maximal T cell responses were achieved, indicating that B cells of all sizes are capable of being regulated in their antigen presentation function through their surface Ig. In addition, B cells of each subpopulation responded to soluble factors present in the supernatants of activated T cells as evidenced by an increase in volume and by the uptake of [3H]thymidine. These results indicate that B cells, regardless of size, are able to participate in at least two essential phases of T cell‐dependent antibody responses, initiating the interaction by processing and presenting antigen to helper T cells and responding to soluble helper factors secreted by activated T cells.
Modification of lymphocyte migration by sulfated polysaccharides
Authors: Brenan Mary , Parish Christopher R.
Published: February 2007

The role of sulfated polysaccharides in lymphocyte migration has been analyzed in vivo using lymphocytes labeled with an intracellular DNA‐binding fluorochrome Hoechst 33342. The influence of a panel of sulfated polysaccharides on entry (by injecting the sulfated polysaccharide prior to the labeled cells) and displacement from lymphoid organs (by injecting the sulfated polysaccharide after the labeled cells have localized) indicated that different sulfated polysaccharides have selective effects on entry and displacement, and furthermore positioning of subpopulations within organs. Additional experiments suggested that receptors for sulfated polysaccharides on high endothelial venules may interact with complementary structures on lymphocytes. The data supporting this conclusion were: (a) the normal localization behavior of lymphocytes preincubated with sulfated polysaccharides; (b) an inverse relationship between the expression of lymphocyte surface receptors for sulfated polysaccharides and the ability of the lymphocytes to enter lymphoid organs and (c) the selective binding of sulfated polysaccharide‐coupled fluoresceinated beads to high endothelial venules. In this case only the beads coupled with the sulfated polysaccharides that inhibited entry bound to the high endothelial venules. These findings are discussed in terms of a fundamental cellular recognition system utilizing sulfated polysaccharides.
Inability of autoimmune mice with the lpr gene to spontaneously produce interleukin 3
Authors: Kelley Vicki E. , Farber Neal M. , Williams John M. , Strom Terry B.
Published: February 2007

A recent report (Palacios, R., Eur. J. Immunol. 1984. 14: 599) indicates that spleen cells from MRL‐MpJ‐lpr/lpr mice spontaneously produce interleukin 3 (IL3) and suggests that this lymphokine could be responsible for promoting the lymphoid hyperplasia regulated by the lpr gene. The present study shows that splenocytes from several murine strains bearing the lpr gene do not spontaneously produce IL3. This has been determined by measuring the amount of this lymphokine by a bioassay and by the detection of IL 3 mRNA using Northern blot analysis. We suggest that another lympbokine is responsible for growth of lymphocytes regulated by the lpr gene.
Immunologic memory to phosphorylcholine (PC) VIII. Expression of the VH−12 gene product in the response to PC‐keyhole limpet hernocyanin
Authors: Rittenberg Marvin B. , Glanville Robert W. , Aebersold Ruedi H. , Chang Sandra P. , Brown Mckay
Published: February 2007

Three murine anti‐phosphorylcholine (PC) hybridomas with group II‐like fine specificity patterns isolated during a memory response to PC‐keyhole limpet hemocyanin (KLH) are examined at the molecular level to determine the origins of the VH and VL used by these antibodies. Southern blots of Hind III cut DNA were hybridized with a probe specific for the V1 gene of the T15 VH family. The V1 germ‐line configuration is retained in these hybridomas indicating that this gene which encodes the VH gene product expressed by most group I anti‐PC hybridomas is not used for antibody production. Southern blots of Eco RI cut DNA hybridized to a probe specific for JH1‐JH4 indicated that all three hybridomas PCG1‐2, PCG1‐3 and PCM‐23 share a 5.2‐kb rearranged JH band, suggesting utilization of a common VH gene segment. N‐terminal amino acid sequence analysis of the heavy chains of two of the hybridoma proteins PCG1‐2 and PCG1‐3 indicates that they belong to mouse heavy chain subgroup II and are closest in sequence to a VH−12 isotype anti‐PC hybridoma protein, HPC‐104, derived from BALB/c mice suppressed for the T15 idiotype; PCG1‐2 and PCG1‐3 each differed from HPC‐104 at only 1/20 residues. In addition, these proteins have in common a lysine at position 1 which has not been found previously in 203 other heavy chain sequences reported. N‐terminal sequences of the light chains of PCG1‐2 and PCG1‐3 are each shown to differ at only 1/22 residues from Vx24, and PCM‐23 had previously been shown to use Vx8; both of these light chains have been associated previously with heavy chains derived from the V1 gene in anti‐PC antibodies.

These results indicate that the VH−12 isotype can be used during a normal antibody response to PC and thus that heavy chains derived from both subgroup II and subgroup III (the T15 heavy chain) contribute to the molecular heterogeneity observed in memory responses to PC‐KLH.
Human leukocyte function‐associated antigens on lympho‐hemopoietic precursor cells
Authors: Campana Dario , Sheridan Brian , Tidman Nicholas , Hoffbrand A. Victor , Janossy George
Published: February 2007

The expression of human leukocyte function‐associated antigens (HLFA, equivalents of murine LFA‐1) was studied on early lympho‐hemopoietic cells in infant thymus and normal bone marrow by double immunofluorescence methods, cell sorting and colony‐forming assays. Monoclonal antibodies MHM‐24 recognizing the α chain (180 kDa) and 60.3 and 60.1 identifying the β chain (95 kDa) were used. The vast majority of thymocytes including large terminal deoxynucleotidyl transferase (TdT)‐positive blast cells are HLFA positive. On the other hand, most B cell precursors in the bone marrow, identified by the expression of nuclear TdT, do not show surface HLFA which appears at the pre‐B cell stage (cytoplasmic μ positive, surface Ig negative). Cell sorting experiments revealed an enrichment of early myeloid cells (myeloblasts and colony‐forming unit cells) in the HLFA‐negative fraction. Erythroid cells appeared to be completely negative from the burst‐forming unit erythrocyte stage onwards. Anti‐HLFA‐antibodies, capable of blocking T cell and natural killer function, may have therapeutic potential without interfering with precursor cell development in man.
Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H‐2‐restricted anti‐HLA T killer lymphocytes
Authors: Achour Ammar , Begue Bernadette , Gomard Elisabeth , Paul Pascale , Sayagh Brigitte , Van Pel Aline , Levy Jean‐Paul
Published: February 2007

The lysis by human and murine anti‐HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK− and P815‐HTR‐TK−. Weak but significant HLA‐A11‐specific lysis was found occasionally with human CTL on the HLA‐A11+ L cells. On the contrary, P815‐A11 or P815‐A2 cells were lysed strongly and specifically by HLA‐A11 or HLA‐A2‐specific human CTL. The T8+T4− phenotype of the effector cells was confirmed and the reaction was inhibited by anti‐HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815‐HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human β2 microglobulin was irrelevant. Anti‐human LFA‐1 antibodies abrogated the lysis of P815‐A11+ cells showing that the LFA‐1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti‐HLA CTL have been prepared by immunizing mice against syngeneic HLA‐A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA‐A11 target cells. This barrier was apparently due to the H‐2 restriction of these H‐2k anti‐HLA murine CTL, as shown by their inability to lyse allogeneic H‐2d cells expressing HLA‐A11, and by the blocking of their activity by anti H‐2k antibodies. By contrast, xenogeneic anti‐HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that (a) human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; (b) the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA‐1 receptor on these cells; (c) a class I molecule of human origin can be seen as an H‐2‐restricted minor histocompatibility antigen in another species.
A model system for the study of the assembly and regulation of human complement C3 convertase (classical pathway)
Authors: Thielens Nicole M. , Colomb Maurice G.
Published: February 2007

The formation of classical C3 convertase of complement and its regulation by C4b‐binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b‐like C4) and iodine‐treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b‐like C4)‐C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P‐glycerol/P‐choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane‐associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle‐associated C3 convertase.
Hapten‐specific cytotoxic T cell clones undergo somatic variation of their antigen recognition specificity
Authors: Weltzien Hans Ulrich , Kempkes Bettina , Jankovic Dragana L. , Eichmann Klaus
Published: February 2007

Two experimental systems have demonstrated somatic variation of antigen recognition specificity of long‐term cytotoxic T cell (CTL) clones. System 1 used CTL clone BT7.4.1 with strict specificity for Kb/TNP, which had been continuously cultured for 15 months in the presence of H‐2b/TNP stimulator cells and interleukin 2. Upon removal of the TNP antigen from the cultures, 99% of the clone cells within about 10 cell divisions lost their ability to grow in the presence of antigen and interleukin 2 (lethal variants). Of the surviving 1%, about 60% retained the ability to lyse target cells in the presence of lectins but only 12% could be considered as “wild type” BT7.4.1 cells, i.e. they still specifically lysed H‐2b/TNP‐bearing target cells. The majority of the growing cells, thus, had to be considered as specificity loss variants. Several specificity loss variants were established in culture and were shown to express membrane‐bound T cell “receptor” heterodimer similar to their TNP‐specific ancester, BT7.4.1. Principally the same types of variants were generated in cultures growing in the presence of TNP antigen, although in quantitatively reduced numbers. Under these conditions the specific stimulator cells appeared to impose a significant selective advantage for “wild type” CTL since even after 15 months the cultures fully retained their specificity for the nominal antigen.

In system 2, the development of cytolytic fine specificity of a panel of 42 individual Kb/TNP‐specific CTL clones was followed over a period of 8 months of in vitro culture. At the beginning of the test, 37 of these clones exhibited significant cross‐reactivity for lysis of H‐2k/TNP target cells. This number of cross‐reactive clones continuously diminished with time and dropped to only 4 clones after 8 months in culture. All 42 clones retained their original Kb/TNP specificity and after losing their reactivity for H‐2k/TNP usually showed no decrease but rather an increase in their cytotoxic activity towards Kb/TNP target cells. Loss of H‐2k/TNP cross‐reactivity was not accompanied by loss of Lyt‐2 or of LFA‐1 surface antigens or by loss of sensitivity of the cytotoxicity to inhibition by anti‐Lyt‐2 or by anti‐LFA‐1 antibody.

We conclude from these observations that in vitro cultivated CTL clones, at least those of C57BL/6 anti‐TNP‐C57BL/6 specificity, are not stable in terms of their antigen recognition specificity. Despite this instability, their nominal cytolytic specificity can be maintained over long periods of in vitro culture and even a maturation to increased antigen specificity is regularly observed, provided antigen is present in the cultures. We assume this indicates that maintenance of clonal specificity in vitro is a matter of antigen‐induced selection rather than a consequence of structural stability of the antigen receptor.
Evidence for a T suppressor cell‐inducing antigenic determinant shared by ADJ‐PC‐5 plasmacytoma and syngeneic BALB/c spleen cells
Authors: Kloke Otto , Haubeck Hans‐Dieter , Kölsch Eckehart
Published: February 2007

The BALB/c plasmacytoma ADJ‐PC‐5 induces in early stages of tumorigenesis specific T suppressor (Ts) cells apparently as the first reaction of the immune system towards the growing tumor. In attempts to apply the concept of early induction of Ts cells in neoplasia for the induction of tolerance to allografts we have tried to induce Ts cells against alloantigens by injecting allogeneic spleen cells in doses increasing exponentially from 10 to 105 cells/mouse. Experiments using various strain combinations failed to demonstrate induction of specific Ts cells by this procedure. To explain the apparent discrepancy between these and the tumor data the capacity of the ADJ‐PC‐5 tumor cells to induce Ts cells in allogeneic combinations was analyzed. ADJ‐PC‐5 cells are capable of inducing specific Ts cells in CBA/J mice. These Ts cells specifically suppress the induction of a primary cytotoxic T cell response of CBA/J spleen cells against BALB/c, BALB.B and (BALB/c × C57BL/6)Fl cells but not one against B10.D2 or C57BL/10 target cells. The ADJ‐PC‐5‐induced CBA/J Ts cells have been further analyzed using a panel of different target or responder cells. From these experiments the following conclusions can be drawn: the CBA/J Ts cells are specific for an antigen which is shared by ADJ‐PC‐5 and normal BALB/c spleen cells. The antigen in question is not coded for by the H‐2d major histocompatibility complex. The data suggest that the Ts cell‐inducing antigen on ADJ‐PC‐5 tumor cells is not a tumor associated neoantigen but a self antigen also present on normal BALB/c lymphoid cells.
Antigen presentation by liposomes: Inhibition with antibodies
Authors: Walden Peter , Nagy Zoltan A. , Klein Jan
Published: February 2007

The involvement of both antigen and class II major histocompatibility complex (MHC) molecules in T cell activation by liposome‐bound antigens was investigated. We used a pigeon cytochrome c (PCC)‐specific Ek‐restricted T cell hybridoma that can be activated to produce interleukin 2 by liposomes carrying either PCC and Ek molecules, or a high concentration of PCC alone. We demonstrated that the MHC‐restricted response of this hybridoma to liposomes is specifically blocked by both anti‐MHC and anti‐PCC monoclonal antibodies, whereas unrestricted activation is only inhibited by PCC‐specific antibody.
Inhibition and enhancement of in vitro helper activity of apocytochrome c‐specific murine T cell populations and clones by anti‐apocytochrome c‐specific monoclonal antibodies
Authors: Titus Richard G. , Corradin Giampietro
Published: February 2007

A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c‐ specific T cell clones and populations in a hapten (trinitropheny1)‐carrier (apocytochrome c) system of T‐B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti‐apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen‐presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen‐ presenting cells.
Interferon‐α/β enhances the expression of Ly‐6 antigens on T cells in vivo and in vitro
Authors: Dumont Francis J. , Coker Lisa Z.
Published: February 2007

Interferons (IFN) have been shown to modulate the expression of a number of cell surface antigens on macrophages and lymphocytes. Because such phenotypic alterations may be involved in the functional effects of IFN, it appears important to characterize further these alterations. In the present work, we evaluated the response to IFN of Ly‐6‐encoded molecules on murine T cells. Two types of Ly‐6 antigens, Ly‐6A and Ly‐6C, normally present on minor subsets of mature T cells were studied. It was found that in vivo treatment of mice with the IFN inducer poly(I.C) or with purified IFN‐α/β resulted two days later in augmented expression of these antigens. Purified T cells cultured in vitro for 2 days in the presence of 5% serum from poly(I.C)‐treated mice or of lo4 units/ml IFN‐α/β also displayed dramatically increased (4‐12‐fold) amounts of Ly‐6 antigens. Under the same conditions, the T cell markers Thy‐1, Ly‐1, Lyt‐2 and MT4 were unaffected or slightly diminished while surface expression of H‐2 or β2‐microglobulin antigens was increased by only 10‐36%. Therefore, poly(1. C)‐ induced or purified IFN interacts with resting T cells to selectively enhance Ly‐6 antigen expression. This phenomenon was found to correlate functionally with increased proliferative response of the T cells, in presence of phorbol myristate acetate, to anti‐Ly‐6 antibodies cross‐linked on their surface. Enhancement of Ly‐6 antigen expression on T cells may thus play a role in IFN‐mediated immunoregulation.
B cell growth and differentiation factors interact with receptors distinct from the interleukin 2 receptor
Authors: Kehrl John H. , Grove Julia H. , Goldsmith Paul K. , Fauci Anthony S.
Published: February 2007

Human B lymphocytes preactivated with Staphylococcus aureus Cowan strain I can proliferate and differentiate to Ig‐secreting cells when cultured in the presence of recombinant interleukin 2 (IL2). We have compared 2 different B cell growth factors (BCGF) and a B cell differentiation factor (BCDF) to IL2 in the regulation of human B lymphocyte growth and differentiation. Utilizing a competitive binding assay, neither a high molecular weight BCGF (HMW‐BCGF) nor a low molecular weight BCGF (LMW‐BCGF) competitively inhibited the binding of radiolabeled IL2. Blocking studies with the anti‐Tac monoclonal antibody demonstrated that B cell proliferation to IL2 was inhibited while a crude supernatant containing BCGF and IL2 was only partially inhibited. B cell Ig secretion induced by IL2 was also inhibited by anti‐Tac while a crude supernatant and partially purified BCDF were not. Furthermore, IL2 plus BCGF was shown to enhance B cell proliferation better than either factor alone and IL2 plus a BCDF enhanced B cell Ig secretion better than either factor alone. By separating activated B cells into Tac‐positive and Tac‐negative fractions, much of the previously noted enhancement with the 2 factors was found to be secondary to the differential sensitivity of the 2 populations to BCGF and IL2 or BCDF and IL2. Thus, LMW‐BCGF, HMW‐BCGF and a partially purified BCDF appear to interact with receptors distinct from the IL2 receptor in mediating their effects on B cell growth and differentiation.