- Email: [email protected]
Accessory Cell-Lymphocyte Interactions
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Accessory Cell-Lymphocyte Interactions Central Lab. of the Netherlands Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunology of the Univ. of Amsterdam, The Netherlands
Human endothelial cell culture supernatant (HECS) contains (a) factor(s) binding to, and promoting the growth of hybridoma cells GIULIA ASTAlDl, MARIA JANSSEN, CH. Wn.tEMS
and
W. P. ZEIJl.EMAKER
The growth of hybridoma cells requires the presence of feeder ceils, especially when low numbers of cells arc cultured and the hybridoma cells arc not fully adapted to the cu lture conditio ns . We found that human endothelial cells as well as their supernatants (HECS) have a growth-promiting activity for hybridoma cells, which is superior to that of ..:conventionaL. feeder cells, such as spleen cells, thymocytes and macrophages. Addition of HECS strongly increases the yield of mouse-mouse and human-mouse hybridoma cells after fusion. Further~ more, HECS can substitute for feeder cells in cloning by limiting dilution of hybridoma cells suggesting that the support to the growth of hybridoma cells is due to soluble factors, not to a celJ-to~cell contact . The proliferation of cultured hybridoma cells , as measured by }Hthymidine incorporation, is strongly enhanced by HECS. HECS also increases the stability of xenogeneic hybridomas between human lymphocytes and mouse myeloma cells, allowing prolonged survival and production of human immunoglobulins. HECS activity can be adsorbed to hybridoma and myeloma cells, but not to other cells such as mouse thymocytes. Moreover, the growth of hybridoma cells is similarly enhanced by a 24 h «pulse» with HECS as in i t.~ continuous presence during culture , indicating that the effect is probably based on delivering a growth~promoting signal to the cells. The production of HECS by endothelial cells is linear in time for 3 days, and is inhibited by the addition of cycloheximide in the culture o~ endothelial cells.
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium
Modulation of immune functions by monoclonal anti-carbohydrate antibodies P.
De
BAETSElIEK, R. HA~lERS,
and j.
GKOOTEN
H ybridoma~derived monoclonal anti~micrococcus antibodies with carbohydrate specificity (galactose, N-acetyl~glucoseamine, N-acetyl-muramic acid) were tested in different «in vitro» immune reactio ns such as T cell proliferative responses to antigen, mitogens and alloantigens and plaque-forming cell (PFC) responses to sheep red blood cells. Different antibodies showed selectivity in modulating a particular .. in vitro» immune response and for at least one function (antigen induced PFC) they did so by interacting at different levels of the immune response. One antibody (clone - C8) showed a broad spectrum of action, interfering with every function tested except for the TgM PFC. This antibody acts at the level of the antigen presenting cell as could be concluded from the inhibition of T cell proliferation and I gG PFC induced through antigen presenting cells (APC). Interfercnce at the level of the APC was also found with two other monoclonal antibodies (elone - D25 and B16). However, in th is case their action was limited to IgG PFC induction si nce no influence was observed on the induction of proliferative T cells. Clone-II H 11 inhibited IgG PFC formation through elimination of a sub~popu lation of non~adherent cells in the presence of complement. A rather specific antibody was clone-AS
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which interfered only with Can A induced proliferation without affecting any other .. in vitro» function. At the present stage of investigation [he exact level of interaction of these monoclonal anti-bacterial antibodies is not yet defined. However, the present data suggest that such antibodies can be used as potent probes for the study of the role of membrane glycoproteins in vanom Immune functions as well as for the identification of various lymphoid cell subpopulations.
Dept. of Medicine, UCLA; Veterans Admin. Hospital , Sepulveda, California, USA
Dependence of mitogen-induced early events on accessory cells in human T lymphocyte growth W. BR USZEWSKI, N. COSH A, L. WYNSTOCK and R.
L ABBE
Mitogen action on T cell~ in vitro is followed by structural and metabolic changes (early everw;) that culminate in DNA replication. Phagocytic and adherent acee.~.<;ory cells (AC) are required for initiation of DNA replication per se, but it is not we1f known whether AC are required for pareicular mitogen-induced early events that precede DNA replica cion. Are AC required for stimulation of glycolysis or ribosomal RNA (rRNA) synthesis - events that precede DNA replication by hours but are essential to it? We propose that the sequence of early events leading to DNA replication is divided into two phases: 1) an early activated seate, comprising certain early events stimulated by mitogen action directly on the T cell and independent of AC; 2) alater, AC-dependenr state, in which mitogen induction of early event.~ requires AC. A recent report (1) supports this hypothesis indirectly. We have reported data directly supporting this hypothesis. We now present simultaneous m ~ asur~ments of single early events in two autologous cell populations: 1) T-enriched leucocytes depleted of AC to give almost complete depression of DNA replication; 2) non-depleted control leucocyte~ which replicate DNA normally. Cells were given a 3D-min exposure to neuraminadase plus galactose oxidase (NG) terminated by effectively complete removal of en7Tmes. Glycolysis wa~ similarly stimulated in both cell populations during the first l2 h of culture. Stimulation of energy metabolism appears to be part of the early activated state, since AC are not required for normal stimulation. Our report of AC-independent stimulation of glucose influx supports this (2). We srudied rRNA synthesis by measuring absolute amounts of unlabeled rRNA species. Synthesis of all species of mature rRNA was stimu lated by NG during the first 20 h in only the non-depleted population. Mitogen stimulation of rRNA synthesis is apparently AC-dependent. Previous data shows that stimulation of gross RNA and protein synthesis is ACdependent (2 ). Most DNA replication was blocked by actinomycin D (0.03 ).tg/ml) inhibition of rRNA ~ynthesi~, indicating the importance of this event to proliferation. Examination of lymphocyte ultrastructural changes of functional significance in the two populations yielded essentially no ob~crvations of AC-independent changes pointing to metabolic events in the early activated state. 1. RESCH, K. , and D. GEMSA. 1979. Immunobiol. 156: 509. 2. BRlJSZEWSKI et al. 1980. Exp. Cell Res. In Press.
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Laboratory for Cellular Immunology, Institu te fo r Biological Research and In stitute of Microbiology and Immunology, School of Medicine, Belgrade, Yugoslavia
Interleukin 1 and 2-Mercaptoethanol can replace macrophages in the activation of suppressor T cells with concanavalin A L. EJDUS-KoNSTA NTI NOVIC and M. M. S1M1C
Unfractionated rat spleen cells prein cubated in primary cultures fo r 2days wi th ConA (5 or 10 f.lg/ ml), then washed with MAG, X-irradiated with 2000 R and mixed I : 1with syngeneic fresh spleen cells in secondary c ultures, inhibited the PHA- or ConA -induced proliferation of the lattcr cells. When , however, not unfractionated but purified splenic T cells depleted of nylon-wool adherent cells were used in preincubation, inhibition did not occur. Instead, an enhanceme nt of both mitogen-induced and spontaneous proliferation of responders in secondary cultures was secn. These findings indicated. on one side, th:n Co nA induces both suppressor and helper cells a nd, on the other side, that macroph ages arc required for induction of suppressors while induction of helper T cells by ConA could be macrophage-independent. Subsequent experiments involving supplementation of splenic T ce ll .~ with peritoneal macroph agcs supported the notion that induction of suppressor T cells by ConA is macrophagedependent. Although the precise functions of macrophages in the process of T cell activation are largely unsolved, in some experimental models i t was demonstrated that macrophages can be replaced by t heir products andl or by 2-mercaptoethanol (2-ME). Therefore, we have exami ned whether 2-ME and carrageenan- induced Interleukin I (IL I ) can substitute for macrophages in ou r experimental model. Results obtained have shown that macrophages needed in the induction of suppressor T cells with ConA can be partially replaced either by lL 1 or by 2-MF.; when these agents acted simu ltaneously, they were found to most efficiently substitute for macrophages. Subsequent experiments with suppressors induced with Co nA among purified splenic T cell s in presence of [L 1 and 2-ME have shown that supp ressors must be restimulated with mitogens in secondary cu ltu res and also must be capable of proliferating in the effector phase in order to exhibit suppression; macrophages seem nOt to be required in the effector phase of s\l ppression. Supported by the Republic of Serbia Research Fund, Belgrade.
The Rockefeller University, New York , N.Y. 1002 1, USA
Rat dendritic cells from lymphoid and non-lymphoid tissues regulate T -lymphocyte transformation W. E. f. KLINK ERT, T . W. WONG, and W. E. BOWERS
Transformation of T -lymphocytes induced by periodate as treatment requires the participation of accessory cells. Recently, we reported that highly purified dendritic cells isolated from lymph nodes function as accessory cells for mitogen response of T-lymphocytes (KL INKI::RT et al. . 1980, PNAS 77 : 54 14). Morphologically similar cells have now been pu rifi ed from spleen, thymus, and normal peritoneal exudate. Moreover, afte r protea.~e di2estion of liver and ~kin preparations enriched in dendritic cells could be obtained fro m these tissues. Dendritic cells from all sources were fo und to differ fro m macrophages in being, non-ad herent, non phagocytic and Fe-receptor negati ve. These cells are radi oresistant, express la-antigens o n thei r
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surface, and rcpre.~ent only 0.01-0 .1 % of the cells present in the starting suspensions. They serve as accessory cells in oxidative mitogenesis of T-Iymphocytes, as potent stimulator cells in syngeneic or allogeneic mixed leukocyte c u lture.~ or in restimulation experiments. Although the kinetic of these reactions is different, the magnitude is dependent upon the number of dendritic cells present in the culture. In contrast to dendritic cells adherent and Fe-receptor positive macrophages isolated from these tissues had no effect in any in vitro assays. Peritoneal exudate macrophages, however, inhibited the accessory cell activity of purified dendritic ce lls from lymph nodes. The mechanism that leads to the activation of T-cells in the presence of purified dendritic cell s is still unknown. Experiments, however, indicate that direct ce ll-cdl interaction might be involved. Dendritic cells separated from mitogen-treated responder lymphocytes by a semipermeable membrane were not able to incluce tra n .~fonnation of cdls. It also appears that la-antigens play a rolc since inclusion of a non-cytotoxic monoclonal antibody, directed aga inst la-antigens, reduced the mitogen response abom 50 % compared to the cOIltrol. Supported by the Deutsche Forschungsgemeinschaft postdoctOral fellowship KL-425 / 1, grant No. IM-240 from the American Cancer Society, and grant PCM-76-166S7 from the National Science Foundation.
The Rockefeller University, New York, N.Y. 10021 , USA
Mitogen responsiveness of rat peripheral blood lymphocytes j. H. LaBADIE. W. E. F. K lI NKERT, and C. F. S HER
Human peripheral blood lymphocytes are frequently used iIl the study of oxidative mitogenesis (blastogenesis induced by sodium periodate or neuraminidase plus galactose oxida!:ie). H owever, we have obtained only low proliferative responses fo llowing treatment of rat peripheral blood lymphocytes with these reagent.~ and have confirmed the responsiveness of human cells. It is unlikely that prostaglandin synthesis by monocytes was inhibitory for the rat cells as indomethacin failed to augment their respome. Our studies on the mechanism of oxidative mitogenesis of rat lymphocytes have led to the identification of an obligatory accessory cell which has a dendriti c morphology (KUNKERT et aI., 1980, P.N.A.S. 77: 5414) and which is not a macrophage. Further, it was determined that dendritic cells were involved in the production of a soluble, interleukin 2-Jike factor which could substitute for the accessory cell. Dendritic accessory cells were subsequently found in a nu mber of rat tissues including lymph nodes. spleen, thymus, peritoneum, liver, and epidermis. Thus, Lewis rat peripheral blood lymphocyte preparations were examined and were found to lack accessory cell activity. Moreover, dend ritic cells could not be isolated from rat blood using techniques suitable for the other tissues . Rat peripheral blood lymphocytes are not inherently unresponsive. Their proliferative response could be restored by the addition of interleukin 2 containing supernates or by the add ition of lymph node dendritic cells. Our results suggest that dendritic cells are present in rat blood in very low numbers and that this may be distinct from d1e human circulatory system. Supported by USPHS AI-1 5867, OFG KL-425/1, and the I. T. H irsch i TrusL
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Department of Pathology, M.D. Anderson Hospital, Houston, TX, Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC, USA
Kinetics of human T-cell proliferation and the role of interleukin 1 A.
MAIZH.
S.
MEHTA,
R.
FORD,
and L.
LACHMAN
The kinetics of T-cell eHJ-Tdr incorporation were examined in respect to those elements necessary and sufficient for the progression of the activated T-cell through the S-phase of the cdl cycle. Alteration in the order of presentation of the monocyte and the lectin to a lymphocyte led to a shift in eHl-T dr incorporation kinetics and indicated there were periods of time early in course of T-cell proliferation when a monocyte was unnecessary. This phenomenon was demonstrated in experiments where short periods (3 h) of lectin-lymphocyte preincubation prior to monocyte exposure eventuated in a maximal DNA synthetic response. The lymphocyte activation process, dependent upon lectin exposure, was reflected morphologically by blastogenesis. Yct this activation process did not lead to significant cell cycle progression. Progression of an activated lymphocyte through G 1 into the S-phase of the cell cycle was shown to be dependent upon the presence of a monocyte. The mechanism by which the monocyte continues those events initiated by lectin stimulation involves the synthesis of Interleukin 1. whose production and/or release is also augmented by lectin exposure. Documentation for the role of human monocyte soluble factors in T-cell mitogenesis was tested using lL-l purified by molecular weight fractionation. isoelectric focusing (pH 6.8-7.2), and gel electrophoresis (rf = 0.36 on 7% acrylamide gels). Human IL-t prepared in this manner could not support the long-term growth of cultured T -cells, yet would augment lectin stimulated human thymocyte and human T-cell mitogenesis. The lL-l preparations were shown to possess this lectin augmenting activity at dilutions containing less than 1 ng of measurable protein. The utilization of purified IL-I eventuated in CHJ-Tdr incorporation kinetics very similar to that achieved with an intact macrophage. This similarity in time of cell cycle progression further indicated that monocyte production of IL-t was not a controlling event in the relatively long T -lymphocyte prereplicative phase.
NIH, Bethesda. MD 20205, USA
Monocytes «pre-incubated. with anti HLA-DR sera (la-like) induced suppression of antigen stimulation and pokeweed-mitogen driven
Ig production D. L.
MANN,
A.
MUCH.r..lORE,
S.
BRODER
HLA-DRw antisera detects antigens controlled by the major histocompatibility complex that are expressed preferentially on B cells and monocytes. Addition of anti-DRw sera to mixed lymphocyte and antigen stimulated cultures inhibits immune responses. The lack of response has been attributed to blocking antigen presentation and/or HLA-D stimulating determinants. The following experiments demonstrate that one effect of ami DRw sera is to induce suppressor T cells. Ig synthesis and antigen stimulated lymphocyte culture were used as an in vitro measurement of immune response. Monocytes were isolated from peripheral blood lymphocytes by adherence or percoll sedimentation and pre-incubated with aUo or hetero anti-DRw sera, normal serum or anti-HLA-A sera for 30 min at 37°C. The monocytes were
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washed and added to pokeweed or antigen (SKSD; tetanus toxoid) stimulated lymphocyte cultures. The effect of the pre-incubated monocytes was measured by reverse hemolytic plaque assay and }H thymidine incorporation. Ig synthesis and 3H thimidine incorporation was reduced by 85 % in cu ltures where monocytes pre-incubated with anti DR sera were added. Monocyrcs pre-incubated with anti HLA-A sera had essentially no effect. When T cells, isolated by E-rosetting from 24 hour cultures of ami DRw treated monocytes were added to pokeweed or antigen stimulated cultures significant suppression of response was observed. When monocytes .. pulsed " with F(ab')2 fragments of alia and hetero DRw sera were added to antigen and pokeweed stimulated cultures, no suppression was observed. The results of these studies indicate that anti-DRw sera can exert an immunoregulatory effect by induction of T suppre.~sor cclls.
Institut fur Biol og i .~che Porschung, FuggerstraBe 3, 0-5000 Koln 90, PRG
Dendritic cell (DC) hybridoma action on T lymphocyte proliferation ]. H. P ETERS
Dendritic cells (DC's) may be the primary inducer cells for T lymphocyte growth, stimulated either by antigens or mirogens. Investigation of the detailed DC function is hampered by the rareness of the~e cells and the absence of tumor lines. For this reason, a number of DC hybridomas has been prepared which retained selected and combined DC properties. CBA mouse DC's were prepared according to a recently developed technique (J. H. PETERS. 1980. Immunobio!. 157: 261 ). They demonstrated little or no phagocytOsis, were adherent to hydrophobic surfaces and weakly positive fo r unspecific e~terase (without prior fixation). They were fused with either BW 5147lymphosarkoma cells from AKR mice or with P3 X 63AG8 plasmocytoma cells from BALB/C mice. In order to select for hybridomas approaching the DC phenotype, rhe cells were kept on hydrophobic surfaces (Petri perm, Heraeus) and non-adherent cells were continuously eliminated. By this regimen 14 different adherent lines could be isolated which in part grew very slowly. Some of them have been successfully cloned. The lines differ markedly in their phenoty pe, both morphologically and functionally. Three of the tested lines exhibit a strong inductive capacity on T lymphocyte growth, in the absence and/or presence of mitogen. One of them stains positive with anti Ta serum, tWO are negative. Further data will be given as how far surface markers characteristic of DC's eosegregate with the lymphocyte stimulatory function of DC hybridomas. Supported in part by the .. Deutsche Forschungsgemeinschaft», SfB 74.
'fInst. of General and Expt!' Pathology, Univ. of Vienna; *'fDiv. of Neurochemistry, Inst. for Brain Research, Austrian Academy of Sciences, Vienna, Austria
Is interaction of macrophages with other cells mediated by gangliosides? :fM. R IEDL, 'fO.
FORSTER, and 'f*H. BERNHEIM F.R
Gangliosides (G) have recently been implicated in a number of immunological phenomena. E.g., they seem to be receptors for type II interferon (1), they were correlated with murine
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NK-activity (2), and it was shown that lymphocytes could be stimulated by antiganglioside antibodies (3). Thy 1 and other T-Iymphocyte antigens were also reported to be associated with G and to playa role in immunoregulation (4). Since macrophage.'> (M el» serve as accessory cells in the immune response. either by secreting regulatory substances (LAF = Interleukin 1) or by direct cell contact, the question arises whether such activity can be mediated by an interaction of Mel> with G. As a first step wc studied the binding of G-coated particles to rat alveolar and peritoneal macrophages (Mct». As model particles we used sheep erythrocytes (E) which have been incubated in G-conraining media presumably leading to insertion of G into their membrane. The fo llowing purified C of bovine and human origin were used: G M1 , G M2 , GliB' GDh , G D1b , Gl'lh and GAL (Svennerholm's nomenclature). Binding was observed of E treated with G M2 > G OJb = G llh > GTla • No binding could be induced by incubation of E with G MI , G M ), GAL' Treatment of Gllla-incubated E with V. cholerac neuraminidase abolished binding. The binding could be partially inhibited by sialyllactose, N-acetyl-neuraminic acid (NANA) and by the G-types which were able to induce binding. From the pattern of binding and inhibitions it is concluded that 1. NANA is essential for binding activity 2. G M2 shows the high est binding affinity 3. the effect seems not merely to be associated with increased negative surface charge of the E induced by G. 1. ANKH, H. et aJ. 1980. Proc. Nat!. Acad. Sci. USA 77: 2528. 2. SCHWARTING, G. A., and A. SUMMERS. 1980. j. Immunol. 124: 1691. 3. SELA, B.-A. et al. 1978. Eur. ]. Immuno!. 8: 268. 4. ESSELMAN, W. J., and H. C. MILLER. 1977. J. Immuno!. 119: 1994.
This study was supported by the Fonds zur Forderung der Wissenschaftlichen Forschung, Austria, project Nr. 3940.
Institut fur Virusforschung, DKFZ, and Institut fur Immunologie der Universitat, 6900 Heidelb~rg, FRG
Accessory cells in the mitogenic activation of rat lymphocytes by concanavalin A 1. SCI lOBER, D. G EMSA, and K.
RE~ClI
Mitogen-activated lymphocytes have been shown to usually require the help of accessory cells to enter proliferation. To better define the nature and role of accessory cells. we have depleted rat lymph node lymphocytes from adherent cells by passage over 0.1 mm glass bead columns. Depleted lymphocytes contained less than 0.2 percent cells positive for non-specific esterase or neutral red ingestion. In the presence of serum, normal untreated lymphocytes responded optimally with DNA synthesis at concentrations of 2 X 10" cells/ml after stimulation with concanavalin A (Con A). Depleted lymphocytes could not be activated to DNA synthesis at these cell densities. DNA replication could be restituted by peritoneal exudate macro phages, optimally when 4-S-percent macrophages were added. The proliferative response of depleted lymphocytes could be also restored at least partially, by raising the cell density to 5 X 10" cellslml. In serum free medium, normal (i.e. adherent cell containing) as well as deple\:ed lymphocyted did not enter mitosis when stimulated at low cell densities. i.c. 2 X lOb ceHs/m!. With both cell populations optimal proliferation resulted when the cell densities were raised to 4-5 X 106 cells/mt. Incorporation of 3H thymidine into DNA under these conditions was comparable to that of normal lymph node cells in the presence of serum.
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Thc proliferative response of depleted lymphocytes at low cell densities could also be restituted by adding mitomycin C~treated cells to a final cell density of 4-5 x 106 cells/ml. Irradiated normal (adherent cell containing) lymphocytes reconstituted Con A~induced prolif~ eration in the presence of serum, but failed to do so in the absence of ~crum. Irradiated depleted lymph node lymphocytes were unable to restitute the proliferacion response of depleted lymphocytes in the pre.~ence or absence of serum. The data suggest that at least two populations of accessory cells exist which support DNA replication of Con A stimulated cells a) a radioresistant one which is adherent and requires serum for its activity and b) a radiosensitive one the action of which is independent of serum and which i.~ contained in the nonadherem lymphocyte population. Furthermore, the role of serum in the mitogenic activation of lym phocytes may consist in activating accessory adherent cells. Supported by the Deutsche Forschungsgemeinschaft.
*Developmental and Metabolic Neurology Branch and **the Laboratory of Vision Research, NEl, NINCDS, National Institutes of Health, Bethesda, Maryland 20205, USA
Production and release of lymphocyte activating factor (LA F) by human monocytes and their derived macrophages (M
GER.Y"~'
Level s of intracellular and extracellular LAF were determined in cultures of human monocytes incubated for various intenrals with different agents. During 7 days in culture, the monocytes transformed into full y differentiated macrophages (M ~). Intracellular LAF increased in unstimulated cultures, but more slowly than has been observed for murine peritoneal M~ cultures. Human cells reached a peak after incubation of 24 hrs, versus 4 hrs for murine Mell. Latex beads (5 X 1011ml) increased both the intracellular and extracellular levels of the mediator throughout the tested incubation intervals. Silica particles increased the release of LAF at tested intervals, but elevated the intracellular activity only in the early intervals of incubation. Addition of LPS increased significantly both intra~ and extracellular LAF levels, as soon as the first 4 hrs of incubation when tested during the first day in culture. However, if added after 3 or 7 days of culture, LPS produced an increase in intracellular LAF activity alone. Practically no +:release" was observed at these intervals. This pattern of eHect with LPS resemble~ that found previously with murine peritoneal macrophagcs in which only intracellular LAF was increased. these data show that there is no relationship per se between the production and release of LAF. Further, it i.~ suggested that monocytes ,an be distinguished from differentiated M
Medical Research lmtitute ; Beilinson Medical Center, Petah~Tikva, Israel, and TeI~Aviv University Sackler School of Medicine; and the Rogosin Kidney Center, Departments of Biochemistry and Medicine, Cornell University Medical College, New York, N.Y. 10021, USA
Rogoff~Wellcome
Enucleated human mononuclear cells treated with neuraminidase and galactose oxidase are mitogenic for human peripheral lymphocytes T. SHKOlN IK, A. L. RUBIN, K. H . STJ::NZI:::L, and A. NOVOGRODSKY
Irradiated or mitomycin C treated, neuraminidase and galactose oxidase (NAGO)~modified human mononuclear cells are mitogenic for human peripheral lymphocytes (indirect stimula-
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tion). Indirect stimulation could result, fro m the interaction of aldehyde moieties, generated on the stimulatory cells by the oxidizing agent, with membrane sites on the responding cells. Alternatively, oxidation might trigger the synthesis of new moieties in the stimulatory ceJl , which could be cell bound or released into the medium. The fo rmation of these stimulatory components could be dependent on the synthetic activity of the nucleus. Sonication Or freeze thawing markedly inhibit the stimulatory capacity of these cells. Since the!\e procedures for cell di sruption affect both cell membrane and nucleus, the specific role of these organelJes in the generation of the stimulatory cells cannot be evaluated. We attempted to resolve this problem by studying the st imulatory capacity of enucleated cells. Mononuclear cells were obtained from human peripheral blood by Picoll-Hypaque density gradient centrifugation. Enucleation and separation of enucleates were achieved by centrifugation in discontinuous Ficoll density gradients in the presence of Cytochalasin B. The degree of purity of the enucleated cells was 99 %, as assessed by microscopic analysis. The enucleated cell s were not stimulated by NAGO treatment as measured by(3-H)Leucine and (3 -H)Thymidine incorporation, they maintained, however, a basal level of protein synthesis. Th e enucl eated cells maintained their capacity to produce lymphocyte activation factor (LAF) (78 % of intact cells) and failed to produce T cell growth factor (rCGF) after NAGO stimu lation. NAGO treatment of the enucleated cells renders them mitogenic for human lymphocytes. Their mitogenic activity was 80 % of intact cdls rendered stimulatory by NAGO modificat ion . It is concluded that nuclear events are not associated with the generation of stimulatory cells by NAGO treatment. Furthermore, it is suggested that the integrity of cell membrane is important fo r the ability of the stimulatory cel1 to elic it the mitogenic signal.
Laboratory of Immunology, Dept. of Microbiology, University of U lm, 7900 U lm, FRG
Different helper requirements for PHA-induced colony formation of low and high density human T-lymphocytes A.
J.
UI.MER and H. -D.
FLAD
Prev ious experiments have shown that after density gradient centrifugation of peripheral blood mononuclear cells (PBMC) only cells from low density fractions can be stimulated by PHA to form T-Iymphocytc (T L) colonies in agar cultures (1. Imm . Methods 30: 1,1979). The present study was designed to investigate the role of helper cclls in colony formation of different human T-cell subsets. T-cells isolated by E-rose tting were further !ieparated by discontinuous grad ient centrifugation with Percoll®. Fractions ( F) 2, 3, 4 and 5 contained 0.5 % or less, f 6 5 % and F 7 30 % of monocytes. In most experiments, only cells derived of fractions 4, 5, 6 and 7 formed colonies. Addition of adherent PBMC (> 95 % mo nocytes) resulted in colony formation also of cell s from F 2 and F 3, enhanced colony formation of F-4 and F 5cdls, but had no considerab le effect on F 6 and F 7 cells. Culture supernatants of PH A stimulated PBMC (SUP-PHA) containing interleukin-2 and those of LPS stimulated PBMC ( SUP ~ LPS ) containing interleukin-l could substitute fo r adherent cells in the system. It is shown that high density T-lymphocyte~ need higher numbers of adherent cells or higher concentrations of SUP- PHA o r SU P-LPS fo r the induction of colonies than Jow density Tlymphocytes. From these results we conclude that T . lymphocyte subsets defined by their d ifferent density have di,tinct requirements for helper cdb or helper factors to induce TLcolonies. Supported by Deu tsche Forschu ngsgcmeinschaft, Sonderforschungsbereich 112/ C-4.
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Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
TPA* mimics interleukin-l in the induction of mitogen responsiveness and maturation of immature human thymocytes
J.
E. De VRIES, P. A. VYTH-DREESE , H. SPITS and C. FIGDOR
Immature human thymocytes (IHT) which were unresponsive to mitogens and alloantigens were separated by countercurrent elutriation centrifugation (CRC). The IHT could be induced to respond to phytohaemagglutinin (PHA) after addition of irradiated allogeneic human monocytes purified by CEC, lL-l isolated from supernatants of monocyte cultures activated with TPA, or TPA at concentrations of 1-10 .} IAg/ m!. TPA, which induces T cell proliferation, was not mitogenic for IHT. Phenotyping of the IHT before and after proliferation revealed a strong decrease in the % of rhymocytes reacting with peanut agglutinin and the monoclonal anti-human thymocyte antibodies OKT-6 and MAS-036, while the % of thymocytes reacting with the monoclonal antibodies OKT-I (directed against all T cells and mature thymocytes) and MAS-015 (ami-HLA A, Band C) was increased 3-6 fold. In addition, the fluorescence intensity of the reaction with OKT-I and MAS-OIS was strongly increased after proliferation. Furthermore, 30-50 % of OKT-l + cells were detected in proliferating IHT cultures, initially completely depleted of OKT-l + cells. The lack of proliferation of the IHT was shown to be d ue to the failure of these cells to produce lL-2 in the presence of PHA. In contrast, the IHT did produce IL-2 after the addition of IL-\ or TPA , but no proliferation was observed. Proliferation of the IHT was only induced if IL-l or TPA were added in combination with PHA. Our data indicate that 1. induction of PH A-responsiveness of the IHT is.ll. result of IL-I or TPA induced maturation/ differentiation, 2. activation of the IHT (expression of receptors for IL-2?) by PHA is required for the induction of proliferative responses to IL-2 produced by the IHT in the presence of IL-l or TPA.
*) TPA
=
12-0-tetradecanoyl phorbol-13-acetate
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Accessory Cell-Lymphocyte Interactions Central Lab. of the Netherlands Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunology of the Univ. of Amsterdam, The Netherlands
Human endothelial cell culture supernatant (HECS) contains (a) factor(s) binding to, and promoting the growth of hybridoma cells GIULIA ASTAlDl, MARIA JANSSEN, CH. Wn.tEMS
and
W. P. ZEIJl.EMAKER
The growth of hybridoma cells requires the presence of feeder ceils, especially when low numbers of cells arc cultured and the hybridoma cells arc not fully adapted to the cu lture conditio ns . We found that human endothelial cells as well as their supernatants (HECS) have a growth-promiting activity for hybridoma cells, which is superior to that of ..:conventionaL. feeder cells, such as spleen cells, thymocytes and macrophages. Addition of HECS strongly increases the yield of mouse-mouse and human-mouse hybridoma cells after fusion. Further~ more, HECS can substitute for feeder cells in cloning by limiting dilution of hybridoma cells suggesting that the support to the growth of hybridoma cells is due to soluble factors, not to a celJ-to~cell contact . The proliferation of cultured hybridoma cells , as measured by }Hthymidine incorporation, is strongly enhanced by HECS. HECS also increases the stability of xenogeneic hybridomas between human lymphocytes and mouse myeloma cells, allowing prolonged survival and production of human immunoglobulins. HECS activity can be adsorbed to hybridoma and myeloma cells, but not to other cells such as mouse thymocytes. Moreover, the growth of hybridoma cells is similarly enhanced by a 24 h «pulse» with HECS as in i t.~ continuous presence during culture , indicating that the effect is probably based on delivering a growth~promoting signal to the cells. The production of HECS by endothelial cells is linear in time for 3 days, and is inhibited by the addition of cycloheximide in the culture o~ endothelial cells.
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Belgium
Modulation of immune functions by monoclonal anti-carbohydrate antibodies P.
De
BAETSElIEK, R. HA~lERS,
and j.
GKOOTEN
H ybridoma~derived monoclonal anti~micrococcus antibodies with carbohydrate specificity (galactose, N-acetyl~glucoseamine, N-acetyl-muramic acid) were tested in different «in vitro» immune reactio ns such as T cell proliferative responses to antigen, mitogens and alloantigens and plaque-forming cell (PFC) responses to sheep red blood cells. Different antibodies showed selectivity in modulating a particular .. in vitro» immune response and for at least one function (antigen induced PFC) they did so by interacting at different levels of the immune response. One antibody (clone - C8) showed a broad spectrum of action, interfering with every function tested except for the TgM PFC. This antibody acts at the level of the antigen presenting cell as could be concluded from the inhibition of T cell proliferation and I gG PFC induced through antigen presenting cells (APC). Interfercnce at the level of the APC was also found with two other monoclonal antibodies (elone - D25 and B16). However, in th is case their action was limited to IgG PFC induction si nce no influence was observed on the induction of proliferative T cells. Clone-II H 11 inhibited IgG PFC formation through elimination of a sub~popu lation of non~adherent cells in the presence of complement. A rather specific antibody was clone-AS
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which interfered only with Can A induced proliferation without affecting any other .. in vitro» function. At the present stage of investigation [he exact level of interaction of these monoclonal anti-bacterial antibodies is not yet defined. However, the present data suggest that such antibodies can be used as potent probes for the study of the role of membrane glycoproteins in vanom Immune functions as well as for the identification of various lymphoid cell subpopulations.
Dept. of Medicine, UCLA; Veterans Admin. Hospital , Sepulveda, California, USA
Dependence of mitogen-induced early events on accessory cells in human T lymphocyte growth W. BR USZEWSKI, N. COSH A, L. WYNSTOCK and R.
L ABBE
Mitogen action on T cell~ in vitro is followed by structural and metabolic changes (early everw;) that culminate in DNA replication. Phagocytic and adherent acee.~.<;ory cells (AC) are required for initiation of DNA replication per se, but it is not we1f known whether AC are required for pareicular mitogen-induced early events that precede DNA replica cion. Are AC required for stimulation of glycolysis or ribosomal RNA (rRNA) synthesis - events that precede DNA replication by hours but are essential to it? We propose that the sequence of early events leading to DNA replication is divided into two phases: 1) an early activated seate, comprising certain early events stimulated by mitogen action directly on the T cell and independent of AC; 2) alater, AC-dependenr state, in which mitogen induction of early event.~ requires AC. A recent report (1) supports this hypothesis indirectly. We have reported data directly supporting this hypothesis. We now present simultaneous m ~ asur~ments of single early events in two autologous cell populations: 1) T-enriched leucocytes depleted of AC to give almost complete depression of DNA replication; 2) non-depleted control leucocyte~ which replicate DNA normally. Cells were given a 3D-min exposure to neuraminadase plus galactose oxidase (NG) terminated by effectively complete removal of en7Tmes. Glycolysis wa~ similarly stimulated in both cell populations during the first l2 h of culture. Stimulation of energy metabolism appears to be part of the early activated state, since AC are not required for normal stimulation. Our report of AC-independent stimulation of glucose influx supports this (2). We srudied rRNA synthesis by measuring absolute amounts of unlabeled rRNA species. Synthesis of all species of mature rRNA was stimu lated by NG during the first 20 h in only the non-depleted population. Mitogen stimulation of rRNA synthesis is apparently AC-dependent. Previous data shows that stimulation of gross RNA and protein synthesis is ACdependent (2 ). Most DNA replication was blocked by actinomycin D (0.03 ).tg/ml) inhibition of rRNA ~ynthesi~, indicating the importance of this event to proliferation. Examination of lymphocyte ultrastructural changes of functional significance in the two populations yielded essentially no ob~crvations of AC-independent changes pointing to metabolic events in the early activated state. 1. RESCH, K. , and D. GEMSA. 1979. Immunobiol. 156: 509. 2. BRlJSZEWSKI et al. 1980. Exp. Cell Res. In Press.
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Laboratory for Cellular Immunology, Institu te fo r Biological Research and In stitute of Microbiology and Immunology, School of Medicine, Belgrade, Yugoslavia
Interleukin 1 and 2-Mercaptoethanol can replace macrophages in the activation of suppressor T cells with concanavalin A L. EJDUS-KoNSTA NTI NOVIC and M. M. S1M1C
Unfractionated rat spleen cells prein cubated in primary cultures fo r 2days wi th ConA (5 or 10 f.lg/ ml), then washed with MAG, X-irradiated with 2000 R and mixed I : 1with syngeneic fresh spleen cells in secondary c ultures, inhibited the PHA- or ConA -induced proliferation of the lattcr cells. When , however, not unfractionated but purified splenic T cells depleted of nylon-wool adherent cells were used in preincubation, inhibition did not occur. Instead, an enhanceme nt of both mitogen-induced and spontaneous proliferation of responders in secondary cultures was secn. These findings indicated. on one side, th:n Co nA induces both suppressor and helper cells a nd, on the other side, that macroph ages arc required for induction of suppressors while induction of helper T cells by ConA could be macrophage-independent. Subsequent experiments involving supplementation of splenic T ce ll .~ with peritoneal macroph agcs supported the notion that induction of suppressor T cells by ConA is macrophagedependent. Although the precise functions of macrophages in the process of T cell activation are largely unsolved, in some experimental models i t was demonstrated that macrophages can be replaced by t heir products andl or by 2-mercaptoethanol (2-ME). Therefore, we have exami ned whether 2-ME and carrageenan- induced Interleukin I (IL I ) can substitute for macrophages in ou r experimental model. Results obtained have shown that macrophages needed in the induction of suppressor T cells with ConA can be partially replaced either by lL 1 or by 2-MF.; when these agents acted simu ltaneously, they were found to most efficiently substitute for macrophages. Subsequent experiments with suppressors induced with Co nA among purified splenic T cell s in presence of [L 1 and 2-ME have shown that supp ressors must be restimulated with mitogens in secondary cu ltu res and also must be capable of proliferating in the effector phase in order to exhibit suppression; macrophages seem nOt to be required in the effector phase of s\l ppression. Supported by the Republic of Serbia Research Fund, Belgrade.
The Rockefeller University, New York , N.Y. 1002 1, USA
Rat dendritic cells from lymphoid and non-lymphoid tissues regulate T -lymphocyte transformation W. E. f. KLINK ERT, T . W. WONG, and W. E. BOWERS
Transformation of T -lymphocytes induced by periodate as treatment requires the participation of accessory cells. Recently, we reported that highly purified dendritic cells isolated from lymph nodes function as accessory cells for mitogen response of T-lymphocytes (KL INKI::RT et al. . 1980, PNAS 77 : 54 14). Morphologically similar cells have now been pu rifi ed from spleen, thymus, and normal peritoneal exudate. Moreover, afte r protea.~e di2estion of liver and ~kin preparations enriched in dendritic cells could be obtained fro m these tissues. Dendritic cells from all sources were fo und to differ fro m macrophages in being, non-ad herent, non phagocytic and Fe-receptor negati ve. These cells are radi oresistant, express la-antigens o n thei r
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surface, and rcpre.~ent only 0.01-0 .1 % of the cells present in the starting suspensions. They serve as accessory cells in oxidative mitogenesis of T-Iymphocytes, as potent stimulator cells in syngeneic or allogeneic mixed leukocyte c u lture.~ or in restimulation experiments. Although the kinetic of these reactions is different, the magnitude is dependent upon the number of dendritic cells present in the culture. In contrast to dendritic cells adherent and Fe-receptor positive macrophages isolated from these tissues had no effect in any in vitro assays. Peritoneal exudate macrophages, however, inhibited the accessory cell activity of purified dendritic ce lls from lymph nodes. The mechanism that leads to the activation of T-cells in the presence of purified dendritic cell s is still unknown. Experiments, however, indicate that direct ce ll-cdl interaction might be involved. Dendritic cells separated from mitogen-treated responder lymphocytes by a semipermeable membrane were not able to incluce tra n .~fonnation of cdls. It also appears that la-antigens play a rolc since inclusion of a non-cytotoxic monoclonal antibody, directed aga inst la-antigens, reduced the mitogen response abom 50 % compared to the cOIltrol. Supported by the Deutsche Forschungsgemeinschaft postdoctOral fellowship KL-425 / 1, grant No. IM-240 from the American Cancer Society, and grant PCM-76-166S7 from the National Science Foundation.
The Rockefeller University, New York, N.Y. 10021 , USA
Mitogen responsiveness of rat peripheral blood lymphocytes j. H. LaBADIE. W. E. F. K lI NKERT, and C. F. S HER
Human peripheral blood lymphocytes are frequently used iIl the study of oxidative mitogenesis (blastogenesis induced by sodium periodate or neuraminidase plus galactose oxida!:ie). H owever, we have obtained only low proliferative responses fo llowing treatment of rat peripheral blood lymphocytes with these reagent.~ and have confirmed the responsiveness of human cells. It is unlikely that prostaglandin synthesis by monocytes was inhibitory for the rat cells as indomethacin failed to augment their respome. Our studies on the mechanism of oxidative mitogenesis of rat lymphocytes have led to the identification of an obligatory accessory cell which has a dendriti c morphology (KUNKERT et aI., 1980, P.N.A.S. 77: 5414) and which is not a macrophage. Further, it was determined that dendritic cells were involved in the production of a soluble, interleukin 2-Jike factor which could substitute for the accessory cell. Dendritic accessory cells were subsequently found in a nu mber of rat tissues including lymph nodes. spleen, thymus, peritoneum, liver, and epidermis. Thus, Lewis rat peripheral blood lymphocyte preparations were examined and were found to lack accessory cell activity. Moreover, dend ritic cells could not be isolated from rat blood using techniques suitable for the other tissues . Rat peripheral blood lymphocytes are not inherently unresponsive. Their proliferative response could be restored by the addition of interleukin 2 containing supernates or by the add ition of lymph node dendritic cells. Our results suggest that dendritic cells are present in rat blood in very low numbers and that this may be distinct from d1e human circulatory system. Supported by USPHS AI-1 5867, OFG KL-425/1, and the I. T. H irsch i TrusL
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Department of Pathology, M.D. Anderson Hospital, Houston, TX, Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC, USA
Kinetics of human T-cell proliferation and the role of interleukin 1 A.
MAIZH.
S.
MEHTA,
R.
FORD,
and L.
LACHMAN
The kinetics of T-cell eHJ-Tdr incorporation were examined in respect to those elements necessary and sufficient for the progression of the activated T-cell through the S-phase of the cdl cycle. Alteration in the order of presentation of the monocyte and the lectin to a lymphocyte led to a shift in eHl-T dr incorporation kinetics and indicated there were periods of time early in course of T-cell proliferation when a monocyte was unnecessary. This phenomenon was demonstrated in experiments where short periods (3 h) of lectin-lymphocyte preincubation prior to monocyte exposure eventuated in a maximal DNA synthetic response. The lymphocyte activation process, dependent upon lectin exposure, was reflected morphologically by blastogenesis. Yct this activation process did not lead to significant cell cycle progression. Progression of an activated lymphocyte through G 1 into the S-phase of the cell cycle was shown to be dependent upon the presence of a monocyte. The mechanism by which the monocyte continues those events initiated by lectin stimulation involves the synthesis of Interleukin 1. whose production and/or release is also augmented by lectin exposure. Documentation for the role of human monocyte soluble factors in T-cell mitogenesis was tested using lL-l purified by molecular weight fractionation. isoelectric focusing (pH 6.8-7.2), and gel electrophoresis (rf = 0.36 on 7% acrylamide gels). Human IL-t prepared in this manner could not support the long-term growth of cultured T -cells, yet would augment lectin stimulated human thymocyte and human T-cell mitogenesis. The lL-l preparations were shown to possess this lectin augmenting activity at dilutions containing less than 1 ng of measurable protein. The utilization of purified IL-I eventuated in CHJ-Tdr incorporation kinetics very similar to that achieved with an intact macrophage. This similarity in time of cell cycle progression further indicated that monocyte production of IL-t was not a controlling event in the relatively long T -lymphocyte prereplicative phase.
NIH, Bethesda. MD 20205, USA
Monocytes «pre-incubated. with anti HLA-DR sera (la-like) induced suppression of antigen stimulation and pokeweed-mitogen driven
Ig production D. L.
MANN,
A.
MUCH.r..lORE,
S.
BRODER
HLA-DRw antisera detects antigens controlled by the major histocompatibility complex that are expressed preferentially on B cells and monocytes. Addition of anti-DRw sera to mixed lymphocyte and antigen stimulated cultures inhibits immune responses. The lack of response has been attributed to blocking antigen presentation and/or HLA-D stimulating determinants. The following experiments demonstrate that one effect of ami DRw sera is to induce suppressor T cells. Ig synthesis and antigen stimulated lymphocyte culture were used as an in vitro measurement of immune response. Monocytes were isolated from peripheral blood lymphocytes by adherence or percoll sedimentation and pre-incubated with aUo or hetero anti-DRw sera, normal serum or anti-HLA-A sera for 30 min at 37°C. The monocytes were
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washed and added to pokeweed or antigen (SKSD; tetanus toxoid) stimulated lymphocyte cultures. The effect of the pre-incubated monocytes was measured by reverse hemolytic plaque assay and }H thymidine incorporation. Ig synthesis and 3H thimidine incorporation was reduced by 85 % in cu ltures where monocytes pre-incubated with anti DR sera were added. Monocyrcs pre-incubated with anti HLA-A sera had essentially no effect. When T cells, isolated by E-rosetting from 24 hour cultures of ami DRw treated monocytes were added to pokeweed or antigen stimulated cultures significant suppression of response was observed. When monocytes .. pulsed " with F(ab')2 fragments of alia and hetero DRw sera were added to antigen and pokeweed stimulated cultures, no suppression was observed. The results of these studies indicate that anti-DRw sera can exert an immunoregulatory effect by induction of T suppre.~sor cclls.
Institut fur Biol og i .~che Porschung, FuggerstraBe 3, 0-5000 Koln 90, PRG
Dendritic cell (DC) hybridoma action on T lymphocyte proliferation ]. H. P ETERS
Dendritic cells (DC's) may be the primary inducer cells for T lymphocyte growth, stimulated either by antigens or mirogens. Investigation of the detailed DC function is hampered by the rareness of the~e cells and the absence of tumor lines. For this reason, a number of DC hybridomas has been prepared which retained selected and combined DC properties. CBA mouse DC's were prepared according to a recently developed technique (J. H. PETERS. 1980. Immunobio!. 157: 261 ). They demonstrated little or no phagocytOsis, were adherent to hydrophobic surfaces and weakly positive fo r unspecific e~terase (without prior fixation). They were fused with either BW 5147lymphosarkoma cells from AKR mice or with P3 X 63AG8 plasmocytoma cells from BALB/C mice. In order to select for hybridomas approaching the DC phenotype, rhe cells were kept on hydrophobic surfaces (Petri perm, Heraeus) and non-adherent cells were continuously eliminated. By this regimen 14 different adherent lines could be isolated which in part grew very slowly. Some of them have been successfully cloned. The lines differ markedly in their phenoty pe, both morphologically and functionally. Three of the tested lines exhibit a strong inductive capacity on T lymphocyte growth, in the absence and/or presence of mitogen. One of them stains positive with anti Ta serum, tWO are negative. Further data will be given as how far surface markers characteristic of DC's eosegregate with the lymphocyte stimulatory function of DC hybridomas. Supported in part by the .. Deutsche Forschungsgemeinschaft», SfB 74.
'fInst. of General and Expt!' Pathology, Univ. of Vienna; *'fDiv. of Neurochemistry, Inst. for Brain Research, Austrian Academy of Sciences, Vienna, Austria
Is interaction of macrophages with other cells mediated by gangliosides? :fM. R IEDL, 'fO.
FORSTER, and 'f*H. BERNHEIM F.R
Gangliosides (G) have recently been implicated in a number of immunological phenomena. E.g., they seem to be receptors for type II interferon (1), they were correlated with murine
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NK-activity (2), and it was shown that lymphocytes could be stimulated by antiganglioside antibodies (3). Thy 1 and other T-Iymphocyte antigens were also reported to be associated with G and to playa role in immunoregulation (4). Since macrophage.'> (M el» serve as accessory cells in the immune response. either by secreting regulatory substances (LAF = Interleukin 1) or by direct cell contact, the question arises whether such activity can be mediated by an interaction of Mel> with G. As a first step wc studied the binding of G-coated particles to rat alveolar and peritoneal macrophages (Mct». As model particles we used sheep erythrocytes (E) which have been incubated in G-conraining media presumably leading to insertion of G into their membrane. The fo llowing purified C of bovine and human origin were used: G M1 , G M2 , GliB' GDh , G D1b , Gl'lh and GAL (Svennerholm's nomenclature). Binding was observed of E treated with G M2 > G OJb = G llh > GTla • No binding could be induced by incubation of E with G MI , G M ), GAL' Treatment of Gllla-incubated E with V. cholerac neuraminidase abolished binding. The binding could be partially inhibited by sialyllactose, N-acetyl-neuraminic acid (NANA) and by the G-types which were able to induce binding. From the pattern of binding and inhibitions it is concluded that 1. NANA is essential for binding activity 2. G M2 shows the high est binding affinity 3. the effect seems not merely to be associated with increased negative surface charge of the E induced by G. 1. ANKH, H. et aJ. 1980. Proc. Nat!. Acad. Sci. USA 77: 2528. 2. SCHWARTING, G. A., and A. SUMMERS. 1980. j. Immunol. 124: 1691. 3. SELA, B.-A. et al. 1978. Eur. ]. Immuno!. 8: 268. 4. ESSELMAN, W. J., and H. C. MILLER. 1977. J. Immuno!. 119: 1994.
This study was supported by the Fonds zur Forderung der Wissenschaftlichen Forschung, Austria, project Nr. 3940.
Institut fur Virusforschung, DKFZ, and Institut fur Immunologie der Universitat, 6900 Heidelb~rg, FRG
Accessory cells in the mitogenic activation of rat lymphocytes by concanavalin A 1. SCI lOBER, D. G EMSA, and K.
RE~ClI
Mitogen-activated lymphocytes have been shown to usually require the help of accessory cells to enter proliferation. To better define the nature and role of accessory cells. we have depleted rat lymph node lymphocytes from adherent cells by passage over 0.1 mm glass bead columns. Depleted lymphocytes contained less than 0.2 percent cells positive for non-specific esterase or neutral red ingestion. In the presence of serum, normal untreated lymphocytes responded optimally with DNA synthesis at concentrations of 2 X 10" cells/ml after stimulation with concanavalin A (Con A). Depleted lymphocytes could not be activated to DNA synthesis at these cell densities. DNA replication could be restituted by peritoneal exudate macro phages, optimally when 4-S-percent macrophages were added. The proliferative response of depleted lymphocytes could be also restored at least partially, by raising the cell density to 5 X 10" cellslml. In serum free medium, normal (i.e. adherent cell containing) as well as deple\:ed lymphocyted did not enter mitosis when stimulated at low cell densities. i.c. 2 X lOb ceHs/m!. With both cell populations optimal proliferation resulted when the cell densities were raised to 4-5 X 106 cells/mt. Incorporation of 3H thymidine into DNA under these conditions was comparable to that of normal lymph node cells in the presence of serum.
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Thc proliferative response of depleted lymphocytes at low cell densities could also be restituted by adding mitomycin C~treated cells to a final cell density of 4-5 x 106 cells/ml. Irradiated normal (adherent cell containing) lymphocytes reconstituted Con A~induced prolif~ eration in the presence of serum, but failed to do so in the absence of ~crum. Irradiated depleted lymph node lymphocytes were unable to restitute the proliferacion response of depleted lymphocytes in the pre.~ence or absence of serum. The data suggest that at least two populations of accessory cells exist which support DNA replication of Con A stimulated cells a) a radioresistant one which is adherent and requires serum for its activity and b) a radiosensitive one the action of which is independent of serum and which i.~ contained in the nonadherem lymphocyte population. Furthermore, the role of serum in the mitogenic activation of lym phocytes may consist in activating accessory adherent cells. Supported by the Deutsche Forschungsgemeinschaft.
*Developmental and Metabolic Neurology Branch and **the Laboratory of Vision Research, NEl, NINCDS, National Institutes of Health, Bethesda, Maryland 20205, USA
Production and release of lymphocyte activating factor (LA F) by human monocytes and their derived macrophages (M
GER.Y"~'
Level s of intracellular and extracellular LAF were determined in cultures of human monocytes incubated for various intenrals with different agents. During 7 days in culture, the monocytes transformed into full y differentiated macrophages (M ~). Intracellular LAF increased in unstimulated cultures, but more slowly than has been observed for murine peritoneal M~ cultures. Human cells reached a peak after incubation of 24 hrs, versus 4 hrs for murine Mell. Latex beads (5 X 1011ml) increased both the intracellular and extracellular levels of the mediator throughout the tested incubation intervals. Silica particles increased the release of LAF at tested intervals, but elevated the intracellular activity only in the early intervals of incubation. Addition of LPS increased significantly both intra~ and extracellular LAF levels, as soon as the first 4 hrs of incubation when tested during the first day in culture. However, if added after 3 or 7 days of culture, LPS produced an increase in intracellular LAF activity alone. Practically no +:release" was observed at these intervals. This pattern of eHect with LPS resemble~ that found previously with murine peritoneal macrophagcs in which only intracellular LAF was increased. these data show that there is no relationship per se between the production and release of LAF. Further, it i.~ suggested that monocytes ,an be distinguished from differentiated M
Medical Research lmtitute ; Beilinson Medical Center, Petah~Tikva, Israel, and TeI~Aviv University Sackler School of Medicine; and the Rogosin Kidney Center, Departments of Biochemistry and Medicine, Cornell University Medical College, New York, N.Y. 10021, USA
Rogoff~Wellcome
Enucleated human mononuclear cells treated with neuraminidase and galactose oxidase are mitogenic for human peripheral lymphocytes T. SHKOlN IK, A. L. RUBIN, K. H . STJ::NZI:::L, and A. NOVOGRODSKY
Irradiated or mitomycin C treated, neuraminidase and galactose oxidase (NAGO)~modified human mononuclear cells are mitogenic for human peripheral lymphocytes (indirect stimula-
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tion). Indirect stimulation could result, fro m the interaction of aldehyde moieties, generated on the stimulatory cells by the oxidizing agent, with membrane sites on the responding cells. Alternatively, oxidation might trigger the synthesis of new moieties in the stimulatory ceJl , which could be cell bound or released into the medium. The fo rmation of these stimulatory components could be dependent on the synthetic activity of the nucleus. Sonication Or freeze thawing markedly inhibit the stimulatory capacity of these cells. Since the!\e procedures for cell di sruption affect both cell membrane and nucleus, the specific role of these organelJes in the generation of the stimulatory cells cannot be evaluated. We attempted to resolve this problem by studying the st imulatory capacity of enucleated cells. Mononuclear cells were obtained from human peripheral blood by Picoll-Hypaque density gradient centrifugation. Enucleation and separation of enucleates were achieved by centrifugation in discontinuous Ficoll density gradients in the presence of Cytochalasin B. The degree of purity of the enucleated cells was 99 %, as assessed by microscopic analysis. The enucleated cell s were not stimulated by NAGO treatment as measured by(3-H)Leucine and (3 -H)Thymidine incorporation, they maintained, however, a basal level of protein synthesis. Th e enucl eated cells maintained their capacity to produce lymphocyte activation factor (LAF) (78 % of intact cells) and failed to produce T cell growth factor (rCGF) after NAGO stimu lation. NAGO treatment of the enucleated cells renders them mitogenic for human lymphocytes. Their mitogenic activity was 80 % of intact cdls rendered stimulatory by NAGO modificat ion . It is concluded that nuclear events are not associated with the generation of stimulatory cells by NAGO treatment. Furthermore, it is suggested that the integrity of cell membrane is important fo r the ability of the stimulatory cel1 to elic it the mitogenic signal.
Laboratory of Immunology, Dept. of Microbiology, University of U lm, 7900 U lm, FRG
Different helper requirements for PHA-induced colony formation of low and high density human T-lymphocytes A.
J.
UI.MER and H. -D.
FLAD
Prev ious experiments have shown that after density gradient centrifugation of peripheral blood mononuclear cells (PBMC) only cells from low density fractions can be stimulated by PHA to form T-Iymphocytc (T L) colonies in agar cultures (1. Imm . Methods 30: 1,1979). The present study was designed to investigate the role of helper cclls in colony formation of different human T-cell subsets. T-cells isolated by E-rose tting were further !ieparated by discontinuous grad ient centrifugation with Percoll®. Fractions ( F) 2, 3, 4 and 5 contained 0.5 % or less, f 6 5 % and F 7 30 % of monocytes. In most experiments, only cells derived of fractions 4, 5, 6 and 7 formed colonies. Addition of adherent PBMC (> 95 % mo nocytes) resulted in colony formation also of cell s from F 2 and F 3, enhanced colony formation of F-4 and F 5cdls, but had no considerab le effect on F 6 and F 7 cells. Culture supernatants of PH A stimulated PBMC (SUP-PHA) containing interleukin-2 and those of LPS stimulated PBMC ( SUP ~ LPS ) containing interleukin-l could substitute fo r adherent cells in the system. It is shown that high density T-lymphocyte~ need higher numbers of adherent cells or higher concentrations of SUP- PHA o r SU P-LPS fo r the induction of colonies than Jow density Tlymphocytes. From these results we conclude that T . lymphocyte subsets defined by their d ifferent density have di,tinct requirements for helper cdb or helper factors to induce TLcolonies. Supported by Deu tsche Forschu ngsgcmeinschaft, Sonderforschungsbereich 112/ C-4.
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Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
TPA* mimics interleukin-l in the induction of mitogen responsiveness and maturation of immature human thymocytes
J.
E. De VRIES, P. A. VYTH-DREESE , H. SPITS and C. FIGDOR
Immature human thymocytes (IHT) which were unresponsive to mitogens and alloantigens were separated by countercurrent elutriation centrifugation (CRC). The IHT could be induced to respond to phytohaemagglutinin (PHA) after addition of irradiated allogeneic human monocytes purified by CEC, lL-l isolated from supernatants of monocyte cultures activated with TPA, or TPA at concentrations of 1-10 .} IAg/ m!. TPA, which induces T cell proliferation, was not mitogenic for IHT. Phenotyping of the IHT before and after proliferation revealed a strong decrease in the % of rhymocytes reacting with peanut agglutinin and the monoclonal anti-human thymocyte antibodies OKT-6 and MAS-036, while the % of thymocytes reacting with the monoclonal antibodies OKT-I (directed against all T cells and mature thymocytes) and MAS-015 (ami-HLA A, Band C) was increased 3-6 fold. In addition, the fluorescence intensity of the reaction with OKT-I and MAS-OIS was strongly increased after proliferation. Furthermore, 30-50 % of OKT-l + cells were detected in proliferating IHT cultures, initially completely depleted of OKT-l + cells. The lack of proliferation of the IHT was shown to be d ue to the failure of these cells to produce lL-2 in the presence of PHA. In contrast, the IHT did produce IL-2 after the addition of IL-\ or TPA , but no proliferation was observed. Proliferation of the IHT was only induced if IL-l or TPA were added in combination with PHA. Our data indicate that 1. induction of PH A-responsiveness of the IHT is.ll. result of IL-I or TPA induced maturation/ differentiation, 2. activation of the IHT (expression of receptors for IL-2?) by PHA is required for the induction of proliferative responses to IL-2 produced by the IHT in the presence of IL-l or TPA.
*) TPA
=
12-0-tetradecanoyl phorbol-13-acetate