- Email: [email protected]
C-4: Antigen Presenting Cells
Workshop C-4 Antigen Presenting Cells
St. Louis University School of Medicine, St. Louis, Missouri 63104, USA
231. Defective antigen presentation by uremic macrophages Y. G.
ALEVY
Studies from our laboratory have indicated that rats with experimentally induced uremia have severely suppressed cell mediated immunity (CMI). We have previously demonstrated that uremic macrophages (UM. In the present study we wanted to determine whether the kinetics of Ag presentation, Ag compartmentalization, and Ag processing by UM may also be contributing factors to the suppressed ability of UM to present Ag to T cells. To this end, UM and control M (CM is significantly slower when compared to that of CM. To study the Ag processing capacity of UM we treated CM and UM with chloroquine and monensin, both inhibitors of intracellular events. Our results indicate that there are no significant differences in the effect of these inhibitors on the Ag processing capacities of CM and UM <1>. However, when the effect of pronase treatment on Ag presentation and sequestering by UM was studied, significant differences between UM and CM were observed. Our results indicate that pronase treatment of CM results in a decreased presentation of Ag to T cells relative to pronase's effect upon UM. These results suggest that Ag is sequestered on UM in a manner which is less susceptible to degradation by pronase. This different arrangement of Ag on the cell surface and the slower uptake of Ag by UM may be contributing factors to the inability of UM to present Ag effectively and thus contribute to the suppressed CMI in uremia. Supported by NIH Grant AM27434.
Metabolism Branch, NCI, National Institutes of Health, Bethesda, MD, USA
232. The role of antigen conformation in determining requirements for antigen processing in T-cell activation
J. A.
BERZOFSKY, and H. Z. STREICHER
We studied the difference in requirements for processing and presentation to a single T cell clone of four different forms of the same epitope of sperm whale myoglobin, namely, on the native protein, 2 denatured forms of the protein, or a 22-residue antigenic peptide fragment.
154 . 16th International Leucocyte Culture Conference, Cambridge The T cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys 140. As inhibitors of macrophage processing of antigen we used the lysosomotropic agents, the weak bases chloroquine and NH 4Cl, the cationic ionophore monensin, and the competitive protease inhibitor, leupeptin. When these agents were used to inhibit processing of antigen by presenting cells, and then washed out before T cells were added to culture, they inhibited the presentation of native antigen, but not of fragment (132-;153). To our surprise, the intact but denatured form, S-methyl myoglobin, the same size as native myoglobin, behaved like the fragment, not like the native protein. Apomyoglobin, which is more flexible than native but more nearly retains the native conformation then S-methyl myoglobin, was intermediate in its susceptibility to inhibition. Thus, native myoglobin requires a processing step which appears to involve lysosomal proteolysis, which is not required by fragment (132-153) or the denatured, unfolded forms. The same finding was made for a T cell line specific for a different epitope of myoglobin - an indication that this result is not peculiar to one epitope of myoglobin. For an antigen the size of myoglobin (17,800 MW), it appears that conformational change, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater exposure of hydrophobic residues in the latter which are buried in the core of the native form, we propose that processing may be necessary to expose these hydrophobic residues, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. The fact that the presentation of intrinsic membrane proteins, as in a mixed lymphocyte reaction, is not inhibited by these agents is consistent with this hypothesis.
Department of Anatomy, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.
233. The frequency of mouse spleen cells presenting alloantigens determined by limiting dilution assay
J.
GOODACRE and J. H. ROBINSON
Limiting Dilution Analysis enables the frequency of rare cells to be calculated. Based on these principles we have developed an assay to measure the frequency of antigen presenting cells. We have chosen to study the frequency of cells stimulating mixed leucocyte culture (MLC) and have utilised the response of C57 mouse spleen to C3H mouse allo-antigens in vitro. Our approach has been to derive a population of cells with enhanced reactivity to C3H from primary MLC's of C57 spleen cells against C3H; C3H stimulator cells are then titrated against this population of «responders» in secondary culture. The response is measured by incorporation of tritiated thymidine in «hanging drop" microcultures in Terasaki plates, which have advantages over conventional microwells for this purpose; in particular, the distribution of responding and non-responding wells conforms more closely to a Poisson model. Preliminary data shows a frequency of 1/39,000 for these cells.
Dept. of Surgery, B.M.C., University of Limburg, Maastricht, The Netherlands
234. Endothelial cells as accessory cells for allo-antigens G. GROENEWEGEN and W. A. BUURMAN Accessory cells play an important role in immunological processes. We studied the function of cultured canine endothelial cells as accessory cell for allo-antigens. Mixed lymphocyte
16th International Leucocyte Culture Conference, Cambridge· 155 cultures (M.L.C.) were performed of responder and stimulator populations from peripheral blood, depleted of accessory cells; proliferation and differentiation were measured. Depleted MLC did not result in proliferation and differentiation of lymphocytes. Addition of cultured arterial or venous endothelial cells, originating from the responder dog, led to responses, similar to normal MLC responses. Moreover third party endothelial cells, added to the depleted MLC, also led to normal MLC-responses. It is concluded that cultured endothelial can function as accessory cell in MLC; the function is not MHC (DLA)-restricted.
Dept. of Histology and Cell Biology, Academic Medical Center, Amsterdam, The Netherlands
235. In vitro antigen presentation by murine dendritic cells and
macrophages in ovalbumin-induced T lymphocyte proliferation
M. L. KAPSENBERG, F. STIEKEMA, M. TEUNISSEN, and H. KEIZER Induction of proliferation of helper T lymphocytes in response to soluble antigens requires the participation of antigen presenting cells. We have tested the antigen presenting capacities of dendritic cells (Fc receptor negative and non-phagocytic) and classical macrophages (Fc receptor positive and phagocytic). Varying numbers (up to 3 X 103 per well) of purified spleen dendritic cells or peritoneal macrophages were added to 3 X 105 purified lymph node T cells from ovalbumin (OVA)-primed mice. Addition of higher amounts of dendritic cells resulted in a considerable syngeneic mixed leucocyte reaction (SMLR) that masked the antigen-specific response. In contrast to dendritic cells, macrophages were poor inducers of the SMLR, However, both purified dendritic cells and macrophages appeared to have comparable capacities to induce OVA -specific proliferation of T cells. Further depletion of dendritic cells « 2 %) from the macrophage population, using the anti dendritic antiserum 33 D 1 (kindly provided by Dr. R. M. Steinman), did not affect the antigen presentation by the macrophages. Antigen presentation by dendritic cells and macrophages is synergistic. The response induced by the combination of the populations was almost two times higher than the sum of the responses induced by the individual populations. Although dendritic cells and macrophages are equally effective in presenting OVA to T cells, the mechanisms of handling the antigen are probably different. The lysosomotropic drugs chloroquine and ammonium chloride inhibit presentation of OVA (and KLH) by macrophages but do not inhibit presentation by dendritic cells. Studies with fluorescent antigen show that macrophages rapidly phagocytose antigen, whereas antigen phagocytosis by dendritic cells was not detected. These results suggest that for effective antigen presentation macrophages interiorize and degrade antigen, whereas dendritic cells present native antigen directly on their cell surface.
Imperial Cancer Research Fund, Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, U.K.
236. Accessory cell heterogeneity in anti-mycobacterial responses P. M. KAYE, B. M. CHAIN, and M. FELDMANN The question of heterogeneity of accessory cells (AC) for antibacterial responses has been largely ignored. We have begun to remedy this situation by examining the capacity of various well defined AC populations to stimulate antimycobacterial T cell responses in vitro.
156 . 16th International Leucocyte Culture Conference, Cambridge It has been found that FcR -, Ia + non phagocytic dendritic cells (DC) are more efficient than FcR +, Ia + phagocytic splenic macrophages in presenting not only soluble mycobacterial antigen (as PPD) but, more interestingly, whole mycobacteria. This greater efficiency of DC is expressed both in terms of absolute numbers of cells and in the concentration of antigen required for optimal T cell proliferation. Experiments showed that whole mycobacteria did not breakdown spontaneously to immunogenic fragments during the time course of the assay. The fact that DC are non phagocytic argues against phagocytosis as an obligatory step in the presentation of particulate antigens by all ACs. Peritoneal exudate cells were found to be very poor presenters of whole mycobacteria. Attempts were made to increase their efficiency by culture in y-interferon (y-IFN) containing supernatants. During these studies, it was found that infection with mycobacterium markedly inhibited the usual y-IFN-induced increase in la antigen expression by exudate macrophages. Taken together, these in vitro observations would suggest an important role for DC in the presentation of mycobacterial antigens in vivo. We are currently examining whether similar patterns of AC activity are evident after in vivo antigen pulsing.
Institute of Virus Research, and tLaboratory of Experimental Animal Care, German Cancer Research Center, Heidelberg, FRG
237. T cell activation induced by a soluble mitogen derived from Mycoplasma arthritidis (MAS) H. KIRCHNER, D. GIEBLER, and tW. NICKLAS
Recently a soluble mitogen for mouse spleen cells was isolated from the supernatant of cultured Mycoplasma arthritidis (MAS). As shown by Cole and confirmed by us, this species seems to be the only mycoplasma elaborating the mitogen. MAS is a protein of a molecular weight of about 13 000 and is a strict T cell mitogen. Most remarkable is the immunogenetic control of the reaction in that T cells of certain inbred mouse strains only (e.g. CBA and C3H) are responders. Non-responders include mice of the haplotype H-2b, H-2f, H-2q and H-2s, i.e. the ones that do not express an intact 1-E molecule on the cell surface. The reactivity of spleen cells of responder mice is dependent on accessory cells (AC) and it has been postulated that binding of MAS to AC is the crucial step in T cell activation. We have found that extensive depletion of macrophages by sephadex G-10 columns from spleen cells of CBA mice leads to a marked reduction (about 90 %) of lymphoproliferation. However, the response could be readily restored by the addition of 2-mercaptoethanol to the culture media. In contrast, if CBA spleen cells were depleted of AC by the use of nylon wool columns, there was a complete abolition of the T cell response, but no restoration by 2-ME. Nylon column-treated CBA spleen cells were fully reconstituted by enriched populations of mitomycin C-treated AC. Furthermore, 2-ME was restoring the genetic defect of all non-responder mice to react to MAS (H-2b etc.) Again, in nylon wool treated populations 2-ME was ineffective. Thus, our data suggest that 2-ME is acting on AC and renders them responsive to MAS despite they originally do not express 1-E structures.
16th International Leucocyte Culture Conference, Cambridge· 157 Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, Wiirzburg, FRG
238. Rat bone marrow precursors develop into dendritic accessory cells under the influence of a conditioned medium W. E. F. KLINKERT
Primary in vitro immune responses require the interaction between T lymphocytes and Iapositive antigen-presenting cells. Recently, techniques were established including discontinuous bovine serum albumin gradients, adherence, irradiation, and Fc-rosetting which led to a positive selection of rat dendritic cells and their identification as the major if not the only accessory cell for rat T lymphocytes treated with the mitogen, sodium periodate, or stimulator cells in mixed lymphocyte reactions. Rat dendritic cells which were found to be present in both lymphoid and non-lymphoid tissues have a low density, are non-adherent, radioresistant, nonspecific esterase-negative, Fc-receptor negative and la-positive. Dendritic accessory cells were detected in cultures of low-density bone marrow cells and developed from radiosensitive precursors especially in a medium containing 10-20 % of a supernatant elaborated by Con A-preactivated syngeneic spleen cells. When compared to purified lymph node dendritic cells they were found to be identical in potency and phenotype. Adherent cells from bone marrow possessed neither accessory activity nor influenced the development of dendritic accessory cells. The mature dendritic accessory cells which could be enriched lOOO-fold were no longer radiosensitive. The production of dendritic accessory cells from bone marrow precursors was influenced by the culture medium. Serum components were found to suppress their development. However, semipurified factor(s) released by mitogen-preactivated spleen cells enhanced the number of dendritic cells considerably. Activity resided in substances with a molecular weight in the range of 40,000 Daltons. This material could be separated on a Sephadex G 75 column from rat Interleukin 2 which had no effect on the formation of dendritic cells but did support proliferation of accessory cell-depleted, mitogen-treated lymph node cells.
Clinical Research Centre, Watford Road, Harrow HAl 3UJ, U.K.
239. Antigen presentation by dendritic cells: studies on cells from normal, sensitized or tolerant mice STELLA C. KNIGHT, J. KREJCI, A. GAUTAM, and G. ASHERSON
The role of dendritic cells (DC) in stimulating proliferative responses of lymphocytes in vitro to picrylchloride (PIC) and Oxazalone (OX) has been studied using cells from normal, antigen primed or tolerant mice. Cell suspensions from lymph nodes of CBA mice were used as responder cells and irradiated (2,400 R) syngeneic dendritic cells from mouse lymph nodes were added. Cultures were in 20 !-II hanging drops in inverted Terasaki plates with 2.5-80 x 103 responder cells and 250-4000 DC. Using cells from normal mice as responder cells, adding DC from normal syngeneic lymph nodes caused little or no stimulation. However, stimulation of normal lymphocytes was obtained when the DC added were a) from animals skin-painted one day earlier with PIC or OX or b) were normal DC incubated for 10 minutes in vitro with 0.5 mM PIC. Responder
ISS· 16th International Leucocyte Culture Conference, Cambridge cells from mice sensitized to OX or PIC 6 days earlier gave an enhanced stimulation to DC from mice skin-painted one day earlier with the sensitizing antigen compared to that with DC from mice painted with the other antigen. DC were approximately a hundredfold more effective in producing this stimulation than whole lymph node cells. These results suggest that the level of syngeneic mixed leucocyte reactions can be influenced by the presence of antigen on DC and by the degree of sensitization of the responder animal to that antigen. Some mice were made tolerant using 4 or 5 injections of high doses (3 mg) of picrylsulphonic acid injected intravenously. Lymph node cells from these tolerant animals failed to proliferate with normal DC but responded to stimulation with DC taken from normal lymph nodes 1 day post skin-painting with PIC. A fivefold increase in the numbers of dendritic cells obtained, from the lymph nodes of normal mice, on metrizamide gradients was found one day post skin painting. However, the lymph nodes of tolerant animals skin painted one day earlier did not give this increased yield of DC. Preliminary studies on the DC from PIC-tolerant mice taken one day post skin-painting with PIC showed that they caused little or no stimulation of normal lymph node cells. The possibility that there may be a defect in antigen presentation in tolerant mice will be discussed.
Dept. Cell Biology and Genetics, Erasmus University, Rotterdam and Dept. Infect. Diseases, University Hospital, Leiden, The Netherlands
240. Monoclonal antibody analysis of mouse macrophage cell lines and isolated macrophages P. J. M. LEENEN, P. H. NIBBERING, A. M. A. C. JANSEN, and W. VAN EWIJK
Transformed cell lines are useful tools in the study of macrophage differentiation, because they represent homogenous populations arrested in a certain stage of differentiation. In order to study a possible relationship between surface antigen expression and macrophage differentiation, we analyzed the surface antigen phenotype of a panel of mouse macrophage cell lines (Table), as well as isolated resident and exudate macrophages from peritoneal cavity and lung using monoclonal antisera. The expression of the differentiation antigens Mac-I, -2, -3, F4/S0, T-200, Thy-l and type II Fc receptor (FcR II) was determined quantitatively by means of a sensitive micro ELISA method and flowcytometry. Significant differences in marker phenotype were observed between macrophage cell lines arrested «early» and «late» in differentiation. For example, Thy-l antigen is expressed on immature myeloid cell lines only (viz. RMB-l and WEHI-3), whereas Mac-I, -2, F4/S0 and FcR II are expressed on lines arrested in mature stages. Furthermore, the amount of Mac-I, -2, F4/S0 and FcRIl antigens expressed on the cell surface differs significantly between the different cell lines. Based on these phenotype studies these cell lines can be ordered in a maturational sequence as indicated in the Table. The most differentiated cell line, i.e. WRI9M.l shows a striking similarity in antigenic composition with peritoneal exudate macrophages elicited with thioglycollate; all other cell lines represent more «immature» stages. Furthermore we studied the antigenic composition of various isolated macrophage populations. We showed that the resident peritoneal macrophage population expresses the Mac-I, F4/S0 and FcRIl antigens at a higher level compared to the exudate population. This difference correlates to a more mature differentiational stage of the resident population. A similar difference was observed between resident and BCG-activated alveolar macrophages. Phenotypical comparison of resident alveolar and resident peritoneal macrophages shows that the latter population expresses Mac-I, F4/S0 and FcRIl antigens at a higher level.
16th International Leucocyte Culture Conference, Cambridge· 159 In conclusion, we propose that macrophage differentiation is accompanied by the appearance and increasing density of the Mac-I, F4/80 and FcRII surface antigens, whereas the Thy-l antigen might be a marker for early differentiational stages. Maturation sequence of macrophage cell lines Order
Cell line
2 3 4 5 6 7 8 9 10
Ml RBM-l WEHI-3 WEHI-3B Pu5-1.8 }774-1.6 P388Dl RAW309Cr.1 RAW264.7 WRI9M.1
Department of Anatomy, The Medical School, University of Newcastle Upon Tyne, Newcastle upon Tyne, U.K.
241. Antigen presentation by antigen chemically coupled to tumour cells
J.
H. ROBINSON, A. L.
BENTLEY,
and R. K. JORDAN
We are investigating DNP-ovalbumin (DNP-OA) chemically coupled to tumour cell surfaces as a model system for immune presentation of tumour antigens in vitro. Nylon wool purified T cells from DNP-OA primed mice proliferate in vitro when stimulated with syngeneic spleen cells pulsed with DNP-Ovalbumin (10 7 cells incubated with 100 f.lg DNPOA for 1 h at 37" and washed 3 times) during a 4 day culture period. This response is inhibited by chloroquine. When DNP-OA is chemically coupled to spleen cells (by the chromic chloride and «Woodwards K» method) they also stimulate antigen specific proliferation in DNP-OA primed T cells, but the response is unaffected by chloroquine. Presentation occurs when the dose of antigen used for coupling is below the effective dose for antigen pulsing (1 f.lg/ml) and coupled cells present optimally at lower stimulator cell doses than do pulsed spleen cells. Furthermore EL-4leukaemic cells pulsed with DNP-OA fail to present antigen to primed T cells, but when DNP-OA is chemically coupled to EL-4 cells they stimulate DNPOA specific T cell proliferation inducing responses of the same magnitude as pulsed spleen cells. Thus EL-4 cells are unable to take up and process antigen, but when manipulated to ensure exposure at the cell surface the immune system is capable of recognising an artificial tumour antigen (DNP-OA) on EL-4 cells. We are analysing the Lyt phenotype, possible inducer, cytotoxic or suppressor function and the MHC restriction pattern of T cells responding in this system. The study will also extend to a range of antigens and tumour cell lines.
160 . 16th International Leucocyte Culture Conference, Cambridge University of California at Los Angeles, Los Angeles, CA 90024, U.S.A.
242. Residues not required for receptor recognition strongly affect
antigen presentation to T cell clones
N. SHASTRI, A. MILLER, and E. E. SERCARZ
Hen egg-white lysozyme (HEL) immunization and subsequent in vitro culture allowed selection of I-Ab restricted T cell clones from B10 mice and I_Ad restricted clones from BDFI animals. Upon restimulation, the B10-derived clones optimally proliferate to doses of ringnecked pheasant lysozyme (REL) 100-fold less than the amount of HEL giving a maximal response. This effect was shown to be independent of the fine specificity of the clones. The apparent heteroclicity was maintained when the reduced, carboxymethylated (RCM) lysozymes were compared. In contrast, when the cyanogen bromide cleaved peptides of the RCMlysozymes, L2H and LZR, which have been shown to contain the determinants recognized by all the clones, were compared, there was no preference for the REL-derived product. With the I-Ad restricted clones, a converse situation obtains in that there is no response to REL but equal responsiveness to LZH and LZR. The T cells and a cloned antigen-presenting line can be used in an IL-2 production assay with the same HELREL discrimination. These observed differences must relate to the manner in which the two lysozymes are processed rather than differences in determinants ultimately recognized, and involves regions of the molecules which are not directly involved in T cell recognition. These results indicate that T cell specificity studies based on the usual assays may reflect long-range effects having to do with steps in antigen handling.
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, 1640 St-Genesius-Rode, Belgium
243. Accessory cell-dependent selection of specific T-cell functions in the
rabbit
E. SMET, and P. DE BAETSELIER
We have analysed the accessory role of rabbit lympho-reticular cells (i.e. B cells and macrophages) in the induction and selection of functional rabbit T cells. As a source of accessory cells we used rabbit peripheral blood B cells, purified on anti-Ig petri dishes or adherent rabbit peripheral blood macrophages (M. Indeed, restimulation of antigen-primed lymph node T cells (prepared from surgically removed antigen-primed lymph nodes) with antigen-fed B cells resulted in the induction of potent antigen-specific T helper cells. Antigen-fed M, on the contrary, induced suppressor T cells which inhibit helper activity. These results indicate that, depending on the accessory cell used to present the antigen, different T-cell functions can be selected, namely antigen-fed B cells will signal T-cell proliferation and T-cell help, while antigen-fed M will signal T-cell proliferation and T-cell suppreSSlOn.
16th International Leucocyte Culture Conference, Cambridge' 161 Institute of Virus Research, German Cancer Research Center, Heidelberg, and IDept. of Immunopharmacology, University Med. School. Hannover, FRG
244. Enhancement by indomethacin and analogous drugs of interferon induction by 10-carboxymethyl-9-acridanone in mouse macrophages E. STORCH, H. KIRCHNER, G. MARTINOTTI 1, and D. GEMSA 1
Prostaglandins of the E-type (PGE) often regulate similar processes as interferons (IFN), and inducers of IFN like polyinosinic-polycytidylic acid (poly I:poly C) were reported to stimulate PGE production via IFN in cell cultures. Macrophages are known to be potent producer cells of both IFN and PGE. Therefore we have examined the relationship between production of IFN and PGE in cultures of pure macrophages derived from the bone marrow of C57BL/6 mice using inhibitors of PGE synthesis (indomethacin, carprofen and aspirin). Depending on the dose of 10-carboxymethyl-9-acridanone (CMA) induction of IFN was increased in the presence of inhibitors of cyclooxygenase of which carprofen was most effective. IFN production was up to about hundred-fold enhanced when pure macrophages were incubated with suboptimal concentrations of CMA. The maximal amount of IFN produced upon the optimal dose of CMA, however, was not influenced. The inhibitors of cyclooxygenase themselves did not exhibit IFN-inducing capacity. The IFN response in the presence of the inhibitors of PGE synthesis depended on the type of inducer, since alterations were not observed upon poly I:poly C, while even a decrease occured upon herpes simplex virus. The potentiating effect of indomethacin-like drugs was not only observed in bone marrow-derived macrophages but also in mouse peritoneal exudate cells, spleen cells and fibroblasts. Supernatants of bone marrow-derived macrophages (controls, after stimulation by CMA and/or in the presence of the inhibitors) were tested for PGE content by radioimmunoassay and in all cases levels of PGE were at the borderline of detection. Release of PGE was also absent after phagocytosis of zymosan, an established stimulant of PGE synthesis suggesting the capacity to produce PGE being dimished in bone marrow-derived macrophages. Addition of exogenous PGE to CMA-stimulated macrophages in the presence of the inhibitors of cyclooxygenase did not reverse the enhancement of IFN induction by these drugs. Because of these results we conclude that depending on the inducer different regulatory mechanisms of IFN induction in macrophages exist and that other mechanisms than inhibition of PGE synthesis are responsible for the enhancement of CMA-induced IFN induction by indomethacin and similar drugs.
Cancer institute, Chinese Academy of Medical Sciences, Beijing (Peking), PRC
245. Macrophage activation - Association and dissociation of macrophage cytolytic and cytostatic activities D. SUN and Y. ZHANG
The cytolytic and cytostatic effect (on both in vitro growth of tumor cells and IL2dependent lymphoblasts) of mouse thioglycollate-induced peritoneal macrophages treated with TPA or macrophage-activating factor (MAF) was investigated. The results indicated that: 1) TPA at nanogram dose levels induced macrophage cytostatic activity, but it did not make macrophage cytolytic even though IOOO-fold higher doses were
162 . 16th International Leucocyte Culture Conference, Cambridge used; 2) While optimal doses of MAF could activate macrophages to exert both cytolytic and cytostatic effects on tumor cells, suboptimal doses of MAF activated macrophage cytostatic effect only; even lower dose of MAF was enough for activating macrophages to become suppressive to the growth of ILZ-dependent lymphoblasts; 3) Thioglycollate-induced peritoneal macrophages after in vitro culture for 3-5 days lost their responsiveness to MAF to become cytolytic. However, such macrophages were still capable of being activated by MAF to become cytostatic. Thus, the cytolytic and cytostatic activities of activated macrophages could be both associated and dissociated depending on the nature and dosage of the stimulants and the responsiveness of macrophage.
Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, and IUnit of Microbiology and Immunology, Faculty of Health Sciences, The Ben-Gurion University of the Negev, Beer-Sheva, Israel
246. Heterogeneity of macrophage-hybridomas: relation of distinct functions to structure E. TZEHOVAL, S. SEGALl, N. ZINBERG, and M. FELDMAN We report the generation of macrophage hybridomas, obtained by somatic cell fusion between a macrophage-enriched C3H.eB spleen cell population and a drug-resistant MPC-11 myeloma cell line, designated clone 4TOO.1Ll. Screening for hybridomas possessing macrophage properties was carried out by assaying for the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further study. We found that a clone derived from E2-7 (E2-7.7) did not express Fc receptors but possessed cell surface Ia molecules. In contrast, clones derived from E2-10 (E210.20 and E2-10.50) possessed Fc receptors but lacked Ia molecules. Opsonized erythrocytes were phagocytosed by E2-10.20 and E2-10.50 but not by E2-7.7. Testing the antigenpresenting capacity of the hybridomas by the response of KLH-primed lymph node cells or enriched T lymphocyte populations to KLH-pulsed hybridoma cells, we found that E2-7.7 caused proliferative responses of both lymph node cells and T lymphocytes, whereas E2-10.20 did not. E2-10.S0 could present antigen only to primed lymph node cells (which are composed of T, Band macrophages) but not to primed T cells. We suggest that E2-10.50 interacts thorough an intermediate accessory cell on antigen-primed T cells. This accessory cell was found to be la-positive and plastic adherent. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, since all the clones manifested the same level of KLH-pinocytosis. The segregation of functional properties among the hybridoma clones may help clarify the dependence of distinct functions on defined molecular structures.
Harvard Medical School, Boston, MA, U.S.A.
247. Antigen presentation: Its regulation and mechanisms E. R. UNANUE The presentation of many protein antigens to helper T cells requires that the antigenpresenting cells process the protein in intracellular vesicles. The evidence for intracellular
16th International Leucocyte Culture Conference, Cambridge· 163 processing will be reviewed using as antigen the intracellular pathogenic bacteria Listeria monocytogenes and the small protein hen-egg lysozyme (HEL). Both have to be taken up by the macrophage and internalized, after which the immunogenic determinant shuttles to the cell surface. The portion of HEL presented by the macrophage is contained in a 16 amino acid fragment. Macrophages that have processed the antigen can be fixed and are still able to present it to T cells. Antigen presentation by the macrophage is under careful regulation. This regulation is exercised at the level of expression of Ia molecules. We shall review the evidence for the presence of an amplification circuit in which helper T cells recognize antigen on Iapositive macrophages and release gamma-inteferon that, in turn, recruits macrophages to express Ia and amplifies antigen-presenting cell function. The expression of la by macrophages is under negative control in various tissues, in part, modulated by the level of prostaglandins. In the neonate, there is a paucity of la-positive macrophages resulting from PGE2 and alphafetoprotein. Both these molecules are powerful inhibitors of la expression. The inhibition of la in the macrophage may be one factor to consider to explain immunological unresponsiveness and self-tolerance in the neonate.
Max-Planck-Institut fiir Biologie, Abteilung Immungenetik, CorrensstraBe 42, D-7400 Tiibingen, FRG
248. T cells respond to native antigens: no evidence for the necessity of antigen processing P. WALDEN, Z. A. NAGY, and J. KLEIN
In order to investigate the requirements for antigen presentation to antigen specific major histocompatibility complex (MHC) restricted T helper cells, liposomes were produced containing MHC class II molecules and antigen. It was tested whether the antigen fixed to a membrane that contained the appropriate restriction element is sufficient to activate T cells or whether a molecular alteration of the antigen in a processing step is necessary to create the stimulatory structure. In all cases tested such liposomes were capable of stimulating a proliferative T cell response in the absence of antigen presenting cells. These results show that T cells recognize native antigens and that processing is not an essential step in antigen presentation.
Lehrstuhl fiir Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat Bochum, 4630 Bochum, FRG
249. Identification of differentiation antigens on the murine myeloid/ macrophage cell line Ml by monoclonal antibodies (MABS) U. WILLMER, B. BOENKE, and F. W. FALKENBERG The aim of this study is the characterization of differentiation antigens on macrophage populations with mabs. For this purpose, 2 cell fusions were performed in which spleen cells of DA rats immunized with mouse macrophage populations were fused either with X63-Ag8.653
164 . 16th International Leucocyte Culture Conference, Cambridge mouse (MIV: «interspecies hybridomas») or Y3-Ag 1.2.3 rat (MV: «intraspecies hybridomas,,) myeloma cells. The mabs produced by the hybridomas were characterized by indirect immunofluorescence on various natural cell populations and transformed cell lines. As expected, most of the mabs show extensive crossreactions with several cell lines of lymphoid origin. Some of them did, however, exhibit specificity for macrophages alone or for macrophages and one or two other cell types. The Ml myeloblast cell line turned out to be an ideal object to study the sequential expression of differentiation antigens with the help of these mabs. This cell line was derived from a spontaneous myeloid leukemia of a SL mouse. The cells can differentiate spontaneously into mature macrophages. In this differentiation state they become strongly adherent and exhibit macrophage properties. In addition, differentiation can be induced by various agents, such as: ascitic fluid, supernatant from embryonal fibroblasts (L929), LPS and Con A. Mabs MIV 32, MIV 51, MIV 52, MIV 113 and MIV 116 which show crossreactivity with various lymphoid cell types reacted strongly with the non adherent undifferentiated M1 cells but weakly or not at all with the spontaneously and induced (by ascitic fluid or by L929 supernatant) differentiated adherent M1 cells. In contrast, we observed that the mabs which seem to be specific for macrophages, MV 87, MV 114 and M1 70, reacted with spontaneously as well as with induced differentiated adherent Ml cells, but not with the undifferentiated non adherent cells. Mabs MIV 38 and MIV 55 only reacted with spontaneously differentiated M1 cells. Other agents like LPS and Con A did not change the antigen pattern detected by mabs as compared to the original M1 cells. Thus the sequential appearence of differentiation antigens on cells of different differentiation states can be monitored by our mabs. The antigens recognized are currently being further characterized.
Department of Microbiology and Immunology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan
250. Regulation of immune response by pre administration of cells briefly pulsed with antigen in vitro. III. Induction of antigen specific suppressor cells by UV treated antigen pulsedJ774 cells K. YOKOMURO, A. MABUCHI, O. TSUCHIYA, and Y. KIMURA
The intravenous administration of syngeneic spleen cells (SPCs) briefly pulsed with antigen (Ag) in vitro, results in a profound state of IgE antibody unresponsiveness. This suppression is mediated by two different mechanisms: one of them is responsible for the immediate tolerance which is induced most effectively by antigen pulsed T cells in the subpopulation of SPCs (KOJIMA et ai., 1984) and the other is responsible for the suppression transferred by suppressor cells (YOKOMURO et ai., 1983). To investigate the first step of suppressor cell induction, one of macrophage like cell lines, J774 cells which originated from BALB/c and could not present Ag in vitro to syngeneic primed T cells were pulsed in vitro with DNP-KLH or beef insulin and injected to BALB/c mice (1st recipients). Six days after, SPCs of 1st recipients were transferred to syngeneic mice (2nd recipients). Two-4 hrs later, 2nd recipients were immunized with Ag plus alum. Their IgE levels were measured with passive cutaneous anaphylaxis (PCA) in rats. Following results were obtained: (1) The induction of Ag specific suppressor cells were dependent on the dose of injected Ag pulsed ]774 cells. (2) Ag pulsed ]774 cells exposed to UV light were capable of inducing the immediate tolerance but not capable of inducing the suppressor cells. (3) In some experiments 10 days after the injection of Ag pulsed SPCs, 1st recipients were immunized with Ag plus alum. Four days later, SPCs from 1st recipients were
16th International Leucocyte Culture Conference, Cambridge· 165 transferred to 2nd recipients and then they were immunized. By the immunization before SPCs transfer, Ag pulsed ]774 cells exposed to UV light also could induce suppressor cells. These results suggest that two signals may be necessary for inducing suppressor cells. First one is antigen itself pulsed on UV treated ]774 cells and second one is factors/cells activated by immunization following the transfer of UV treated Ag pulsed cells. KOJIMA, N., A. MABUCHI, K. YOKOMURO, and Y. KIMURA. (1984). Suppression of IgE antibody response by preadministration of antigen-pulsed spleen cells II. char,cteristics of immediate tolerance induction. Int. Archs Allergy app!. Immun., in press. YOKOMURO, K., A. MABUCHI, M. SAIZAWA, N. KOJIMA, A. S. ROSENTHAL, and Y. KIMURA. (1983). Regulation of immune response by preadministration of cells briefly pulsed with antigen in vitro I. suppression of IgE antibody response by antigen pulsed spleen cells. Int. Archs Allergy app!. Immun. 69: 98.
St. Louis University School of Medicine, St. Louis, Missouri 63104, USA
231. Defective antigen presentation by uremic macrophages Y. G.
ALEVY
Studies from our laboratory have indicated that rats with experimentally induced uremia have severely suppressed cell mediated immunity (CMI). We have previously demonstrated that uremic macrophages (UM
Metabolism Branch, NCI, National Institutes of Health, Bethesda, MD, USA
232. The role of antigen conformation in determining requirements for antigen processing in T-cell activation
J. A.
BERZOFSKY, and H. Z. STREICHER
We studied the difference in requirements for processing and presentation to a single T cell clone of four different forms of the same epitope of sperm whale myoglobin, namely, on the native protein, 2 denatured forms of the protein, or a 22-residue antigenic peptide fragment.
154 . 16th International Leucocyte Culture Conference, Cambridge The T cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys 140. As inhibitors of macrophage processing of antigen we used the lysosomotropic agents, the weak bases chloroquine and NH 4Cl, the cationic ionophore monensin, and the competitive protease inhibitor, leupeptin. When these agents were used to inhibit processing of antigen by presenting cells, and then washed out before T cells were added to culture, they inhibited the presentation of native antigen, but not of fragment (132-;153). To our surprise, the intact but denatured form, S-methyl myoglobin, the same size as native myoglobin, behaved like the fragment, not like the native protein. Apomyoglobin, which is more flexible than native but more nearly retains the native conformation then S-methyl myoglobin, was intermediate in its susceptibility to inhibition. Thus, native myoglobin requires a processing step which appears to involve lysosomal proteolysis, which is not required by fragment (132-153) or the denatured, unfolded forms. The same finding was made for a T cell line specific for a different epitope of myoglobin - an indication that this result is not peculiar to one epitope of myoglobin. For an antigen the size of myoglobin (17,800 MW), it appears that conformational change, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater exposure of hydrophobic residues in the latter which are buried in the core of the native form, we propose that processing may be necessary to expose these hydrophobic residues, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell. The fact that the presentation of intrinsic membrane proteins, as in a mixed lymphocyte reaction, is not inhibited by these agents is consistent with this hypothesis.
Department of Anatomy, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K.
233. The frequency of mouse spleen cells presenting alloantigens determined by limiting dilution assay
J.
GOODACRE and J. H. ROBINSON
Limiting Dilution Analysis enables the frequency of rare cells to be calculated. Based on these principles we have developed an assay to measure the frequency of antigen presenting cells. We have chosen to study the frequency of cells stimulating mixed leucocyte culture (MLC) and have utilised the response of C57 mouse spleen to C3H mouse allo-antigens in vitro. Our approach has been to derive a population of cells with enhanced reactivity to C3H from primary MLC's of C57 spleen cells against C3H; C3H stimulator cells are then titrated against this population of «responders» in secondary culture. The response is measured by incorporation of tritiated thymidine in «hanging drop" microcultures in Terasaki plates, which have advantages over conventional microwells for this purpose; in particular, the distribution of responding and non-responding wells conforms more closely to a Poisson model. Preliminary data shows a frequency of 1/39,000 for these cells.
Dept. of Surgery, B.M.C., University of Limburg, Maastricht, The Netherlands
234. Endothelial cells as accessory cells for allo-antigens G. GROENEWEGEN and W. A. BUURMAN Accessory cells play an important role in immunological processes. We studied the function of cultured canine endothelial cells as accessory cell for allo-antigens. Mixed lymphocyte
16th International Leucocyte Culture Conference, Cambridge· 155 cultures (M.L.C.) were performed of responder and stimulator populations from peripheral blood, depleted of accessory cells; proliferation and differentiation were measured. Depleted MLC did not result in proliferation and differentiation of lymphocytes. Addition of cultured arterial or venous endothelial cells, originating from the responder dog, led to responses, similar to normal MLC responses. Moreover third party endothelial cells, added to the depleted MLC, also led to normal MLC-responses. It is concluded that cultured endothelial can function as accessory cell in MLC; the function is not MHC (DLA)-restricted.
Dept. of Histology and Cell Biology, Academic Medical Center, Amsterdam, The Netherlands
235. In vitro antigen presentation by murine dendritic cells and
macrophages in ovalbumin-induced T lymphocyte proliferation
M. L. KAPSENBERG, F. STIEKEMA, M. TEUNISSEN, and H. KEIZER Induction of proliferation of helper T lymphocytes in response to soluble antigens requires the participation of antigen presenting cells. We have tested the antigen presenting capacities of dendritic cells (Fc receptor negative and non-phagocytic) and classical macrophages (Fc receptor positive and phagocytic). Varying numbers (up to 3 X 103 per well) of purified spleen dendritic cells or peritoneal macrophages were added to 3 X 105 purified lymph node T cells from ovalbumin (OVA)-primed mice. Addition of higher amounts of dendritic cells resulted in a considerable syngeneic mixed leucocyte reaction (SMLR) that masked the antigen-specific response. In contrast to dendritic cells, macrophages were poor inducers of the SMLR, However, both purified dendritic cells and macrophages appeared to have comparable capacities to induce OVA -specific proliferation of T cells. Further depletion of dendritic cells « 2 %) from the macrophage population, using the anti dendritic antiserum 33 D 1 (kindly provided by Dr. R. M. Steinman), did not affect the antigen presentation by the macrophages. Antigen presentation by dendritic cells and macrophages is synergistic. The response induced by the combination of the populations was almost two times higher than the sum of the responses induced by the individual populations. Although dendritic cells and macrophages are equally effective in presenting OVA to T cells, the mechanisms of handling the antigen are probably different. The lysosomotropic drugs chloroquine and ammonium chloride inhibit presentation of OVA (and KLH) by macrophages but do not inhibit presentation by dendritic cells. Studies with fluorescent antigen show that macrophages rapidly phagocytose antigen, whereas antigen phagocytosis by dendritic cells was not detected. These results suggest that for effective antigen presentation macrophages interiorize and degrade antigen, whereas dendritic cells present native antigen directly on their cell surface.
Imperial Cancer Research Fund, Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, U.K.
236. Accessory cell heterogeneity in anti-mycobacterial responses P. M. KAYE, B. M. CHAIN, and M. FELDMANN The question of heterogeneity of accessory cells (AC) for antibacterial responses has been largely ignored. We have begun to remedy this situation by examining the capacity of various well defined AC populations to stimulate antimycobacterial T cell responses in vitro.
156 . 16th International Leucocyte Culture Conference, Cambridge It has been found that FcR -, Ia + non phagocytic dendritic cells (DC) are more efficient than FcR +, Ia + phagocytic splenic macrophages in presenting not only soluble mycobacterial antigen (as PPD) but, more interestingly, whole mycobacteria. This greater efficiency of DC is expressed both in terms of absolute numbers of cells and in the concentration of antigen required for optimal T cell proliferation. Experiments showed that whole mycobacteria did not breakdown spontaneously to immunogenic fragments during the time course of the assay. The fact that DC are non phagocytic argues against phagocytosis as an obligatory step in the presentation of particulate antigens by all ACs. Peritoneal exudate cells were found to be very poor presenters of whole mycobacteria. Attempts were made to increase their efficiency by culture in y-interferon (y-IFN) containing supernatants. During these studies, it was found that infection with mycobacterium markedly inhibited the usual y-IFN-induced increase in la antigen expression by exudate macrophages. Taken together, these in vitro observations would suggest an important role for DC in the presentation of mycobacterial antigens in vivo. We are currently examining whether similar patterns of AC activity are evident after in vivo antigen pulsing.
Institute of Virus Research, and tLaboratory of Experimental Animal Care, German Cancer Research Center, Heidelberg, FRG
237. T cell activation induced by a soluble mitogen derived from Mycoplasma arthritidis (MAS) H. KIRCHNER, D. GIEBLER, and tW. NICKLAS
Recently a soluble mitogen for mouse spleen cells was isolated from the supernatant of cultured Mycoplasma arthritidis (MAS). As shown by Cole and confirmed by us, this species seems to be the only mycoplasma elaborating the mitogen. MAS is a protein of a molecular weight of about 13 000 and is a strict T cell mitogen. Most remarkable is the immunogenetic control of the reaction in that T cells of certain inbred mouse strains only (e.g. CBA and C3H) are responders. Non-responders include mice of the haplotype H-2b, H-2f, H-2q and H-2s, i.e. the ones that do not express an intact 1-E molecule on the cell surface. The reactivity of spleen cells of responder mice is dependent on accessory cells (AC) and it has been postulated that binding of MAS to AC is the crucial step in T cell activation. We have found that extensive depletion of macrophages by sephadex G-10 columns from spleen cells of CBA mice leads to a marked reduction (about 90 %) of lymphoproliferation. However, the response could be readily restored by the addition of 2-mercaptoethanol to the culture media. In contrast, if CBA spleen cells were depleted of AC by the use of nylon wool columns, there was a complete abolition of the T cell response, but no restoration by 2-ME. Nylon column-treated CBA spleen cells were fully reconstituted by enriched populations of mitomycin C-treated AC. Furthermore, 2-ME was restoring the genetic defect of all non-responder mice to react to MAS (H-2b etc.) Again, in nylon wool treated populations 2-ME was ineffective. Thus, our data suggest that 2-ME is acting on AC and renders them responsive to MAS despite they originally do not express 1-E structures.
16th International Leucocyte Culture Conference, Cambridge· 157 Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, Wiirzburg, FRG
238. Rat bone marrow precursors develop into dendritic accessory cells under the influence of a conditioned medium W. E. F. KLINKERT
Primary in vitro immune responses require the interaction between T lymphocytes and Iapositive antigen-presenting cells. Recently, techniques were established including discontinuous bovine serum albumin gradients, adherence, irradiation, and Fc-rosetting which led to a positive selection of rat dendritic cells and their identification as the major if not the only accessory cell for rat T lymphocytes treated with the mitogen, sodium periodate, or stimulator cells in mixed lymphocyte reactions. Rat dendritic cells which were found to be present in both lymphoid and non-lymphoid tissues have a low density, are non-adherent, radioresistant, nonspecific esterase-negative, Fc-receptor negative and la-positive. Dendritic accessory cells were detected in cultures of low-density bone marrow cells and developed from radiosensitive precursors especially in a medium containing 10-20 % of a supernatant elaborated by Con A-preactivated syngeneic spleen cells. When compared to purified lymph node dendritic cells they were found to be identical in potency and phenotype. Adherent cells from bone marrow possessed neither accessory activity nor influenced the development of dendritic accessory cells. The mature dendritic accessory cells which could be enriched lOOO-fold were no longer radiosensitive. The production of dendritic accessory cells from bone marrow precursors was influenced by the culture medium. Serum components were found to suppress their development. However, semipurified factor(s) released by mitogen-preactivated spleen cells enhanced the number of dendritic cells considerably. Activity resided in substances with a molecular weight in the range of 40,000 Daltons. This material could be separated on a Sephadex G 75 column from rat Interleukin 2 which had no effect on the formation of dendritic cells but did support proliferation of accessory cell-depleted, mitogen-treated lymph node cells.
Clinical Research Centre, Watford Road, Harrow HAl 3UJ, U.K.
239. Antigen presentation by dendritic cells: studies on cells from normal, sensitized or tolerant mice STELLA C. KNIGHT, J. KREJCI, A. GAUTAM, and G. ASHERSON
The role of dendritic cells (DC) in stimulating proliferative responses of lymphocytes in vitro to picrylchloride (PIC) and Oxazalone (OX) has been studied using cells from normal, antigen primed or tolerant mice. Cell suspensions from lymph nodes of CBA mice were used as responder cells and irradiated (2,400 R) syngeneic dendritic cells from mouse lymph nodes were added. Cultures were in 20 !-II hanging drops in inverted Terasaki plates with 2.5-80 x 103 responder cells and 250-4000 DC. Using cells from normal mice as responder cells, adding DC from normal syngeneic lymph nodes caused little or no stimulation. However, stimulation of normal lymphocytes was obtained when the DC added were a) from animals skin-painted one day earlier with PIC or OX or b) were normal DC incubated for 10 minutes in vitro with 0.5 mM PIC. Responder
ISS· 16th International Leucocyte Culture Conference, Cambridge cells from mice sensitized to OX or PIC 6 days earlier gave an enhanced stimulation to DC from mice skin-painted one day earlier with the sensitizing antigen compared to that with DC from mice painted with the other antigen. DC were approximately a hundredfold more effective in producing this stimulation than whole lymph node cells. These results suggest that the level of syngeneic mixed leucocyte reactions can be influenced by the presence of antigen on DC and by the degree of sensitization of the responder animal to that antigen. Some mice were made tolerant using 4 or 5 injections of high doses (3 mg) of picrylsulphonic acid injected intravenously. Lymph node cells from these tolerant animals failed to proliferate with normal DC but responded to stimulation with DC taken from normal lymph nodes 1 day post skin-painting with PIC. A fivefold increase in the numbers of dendritic cells obtained, from the lymph nodes of normal mice, on metrizamide gradients was found one day post skin painting. However, the lymph nodes of tolerant animals skin painted one day earlier did not give this increased yield of DC. Preliminary studies on the DC from PIC-tolerant mice taken one day post skin-painting with PIC showed that they caused little or no stimulation of normal lymph node cells. The possibility that there may be a defect in antigen presentation in tolerant mice will be discussed.
Dept. Cell Biology and Genetics, Erasmus University, Rotterdam and Dept. Infect. Diseases, University Hospital, Leiden, The Netherlands
240. Monoclonal antibody analysis of mouse macrophage cell lines and isolated macrophages P. J. M. LEENEN, P. H. NIBBERING, A. M. A. C. JANSEN, and W. VAN EWIJK
Transformed cell lines are useful tools in the study of macrophage differentiation, because they represent homogenous populations arrested in a certain stage of differentiation. In order to study a possible relationship between surface antigen expression and macrophage differentiation, we analyzed the surface antigen phenotype of a panel of mouse macrophage cell lines (Table), as well as isolated resident and exudate macrophages from peritoneal cavity and lung using monoclonal antisera. The expression of the differentiation antigens Mac-I, -2, -3, F4/S0, T-200, Thy-l and type II Fc receptor (FcR II) was determined quantitatively by means of a sensitive micro ELISA method and flowcytometry. Significant differences in marker phenotype were observed between macrophage cell lines arrested «early» and «late» in differentiation. For example, Thy-l antigen is expressed on immature myeloid cell lines only (viz. RMB-l and WEHI-3), whereas Mac-I, -2, F4/S0 and FcR II are expressed on lines arrested in mature stages. Furthermore, the amount of Mac-I, -2, F4/S0 and FcRIl antigens expressed on the cell surface differs significantly between the different cell lines. Based on these phenotype studies these cell lines can be ordered in a maturational sequence as indicated in the Table. The most differentiated cell line, i.e. WRI9M.l shows a striking similarity in antigenic composition with peritoneal exudate macrophages elicited with thioglycollate; all other cell lines represent more «immature» stages. Furthermore we studied the antigenic composition of various isolated macrophage populations. We showed that the resident peritoneal macrophage population expresses the Mac-I, F4/S0 and FcRIl antigens at a higher level compared to the exudate population. This difference correlates to a more mature differentiational stage of the resident population. A similar difference was observed between resident and BCG-activated alveolar macrophages. Phenotypical comparison of resident alveolar and resident peritoneal macrophages shows that the latter population expresses Mac-I, F4/S0 and FcRIl antigens at a higher level.
16th International Leucocyte Culture Conference, Cambridge· 159 In conclusion, we propose that macrophage differentiation is accompanied by the appearance and increasing density of the Mac-I, F4/80 and FcRII surface antigens, whereas the Thy-l antigen might be a marker for early differentiational stages. Maturation sequence of macrophage cell lines Order
Cell line
2 3 4 5 6 7 8 9 10
Ml RBM-l WEHI-3 WEHI-3B Pu5-1.8 }774-1.6 P388Dl RAW309Cr.1 RAW264.7 WRI9M.1
Department of Anatomy, The Medical School, University of Newcastle Upon Tyne, Newcastle upon Tyne, U.K.
241. Antigen presentation by antigen chemically coupled to tumour cells
J.
H. ROBINSON, A. L.
BENTLEY,
and R. K. JORDAN
We are investigating DNP-ovalbumin (DNP-OA) chemically coupled to tumour cell surfaces as a model system for immune presentation of tumour antigens in vitro. Nylon wool purified T cells from DNP-OA primed mice proliferate in vitro when stimulated with syngeneic spleen cells pulsed with DNP-Ovalbumin (10 7 cells incubated with 100 f.lg DNPOA for 1 h at 37" and washed 3 times) during a 4 day culture period. This response is inhibited by chloroquine. When DNP-OA is chemically coupled to spleen cells (by the chromic chloride and «Woodwards K» method) they also stimulate antigen specific proliferation in DNP-OA primed T cells, but the response is unaffected by chloroquine. Presentation occurs when the dose of antigen used for coupling is below the effective dose for antigen pulsing (1 f.lg/ml) and coupled cells present optimally at lower stimulator cell doses than do pulsed spleen cells. Furthermore EL-4leukaemic cells pulsed with DNP-OA fail to present antigen to primed T cells, but when DNP-OA is chemically coupled to EL-4 cells they stimulate DNPOA specific T cell proliferation inducing responses of the same magnitude as pulsed spleen cells. Thus EL-4 cells are unable to take up and process antigen, but when manipulated to ensure exposure at the cell surface the immune system is capable of recognising an artificial tumour antigen (DNP-OA) on EL-4 cells. We are analysing the Lyt phenotype, possible inducer, cytotoxic or suppressor function and the MHC restriction pattern of T cells responding in this system. The study will also extend to a range of antigens and tumour cell lines.
160 . 16th International Leucocyte Culture Conference, Cambridge University of California at Los Angeles, Los Angeles, CA 90024, U.S.A.
242. Residues not required for receptor recognition strongly affect
antigen presentation to T cell clones
N. SHASTRI, A. MILLER, and E. E. SERCARZ
Hen egg-white lysozyme (HEL) immunization and subsequent in vitro culture allowed selection of I-Ab restricted T cell clones from B10 mice and I_Ad restricted clones from BDFI animals. Upon restimulation, the B10-derived clones optimally proliferate to doses of ringnecked pheasant lysozyme (REL) 100-fold less than the amount of HEL giving a maximal response. This effect was shown to be independent of the fine specificity of the clones. The apparent heteroclicity was maintained when the reduced, carboxymethylated (RCM) lysozymes were compared. In contrast, when the cyanogen bromide cleaved peptides of the RCMlysozymes, L2H and LZR, which have been shown to contain the determinants recognized by all the clones, were compared, there was no preference for the REL-derived product. With the I-Ad restricted clones, a converse situation obtains in that there is no response to REL but equal responsiveness to LZH and LZR. The T cells and a cloned antigen-presenting line can be used in an IL-2 production assay with the same HELREL discrimination. These observed differences must relate to the manner in which the two lysozymes are processed rather than differences in determinants ultimately recognized, and involves regions of the molecules which are not directly involved in T cell recognition. These results indicate that T cell specificity studies based on the usual assays may reflect long-range effects having to do with steps in antigen handling.
Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, 1640 St-Genesius-Rode, Belgium
243. Accessory cell-dependent selection of specific T-cell functions in the
rabbit
E. SMET, and P. DE BAETSELIER
We have analysed the accessory role of rabbit lympho-reticular cells (i.e. B cells and macrophages) in the induction and selection of functional rabbit T cells. As a source of accessory cells we used rabbit peripheral blood B cells, purified on anti-Ig petri dishes or adherent rabbit peripheral blood macrophages (M
16th International Leucocyte Culture Conference, Cambridge' 161 Institute of Virus Research, German Cancer Research Center, Heidelberg, and IDept. of Immunopharmacology, University Med. School. Hannover, FRG
244. Enhancement by indomethacin and analogous drugs of interferon induction by 10-carboxymethyl-9-acridanone in mouse macrophages E. STORCH, H. KIRCHNER, G. MARTINOTTI 1, and D. GEMSA 1
Prostaglandins of the E-type (PGE) often regulate similar processes as interferons (IFN), and inducers of IFN like polyinosinic-polycytidylic acid (poly I:poly C) were reported to stimulate PGE production via IFN in cell cultures. Macrophages are known to be potent producer cells of both IFN and PGE. Therefore we have examined the relationship between production of IFN and PGE in cultures of pure macrophages derived from the bone marrow of C57BL/6 mice using inhibitors of PGE synthesis (indomethacin, carprofen and aspirin). Depending on the dose of 10-carboxymethyl-9-acridanone (CMA) induction of IFN was increased in the presence of inhibitors of cyclooxygenase of which carprofen was most effective. IFN production was up to about hundred-fold enhanced when pure macrophages were incubated with suboptimal concentrations of CMA. The maximal amount of IFN produced upon the optimal dose of CMA, however, was not influenced. The inhibitors of cyclooxygenase themselves did not exhibit IFN-inducing capacity. The IFN response in the presence of the inhibitors of PGE synthesis depended on the type of inducer, since alterations were not observed upon poly I:poly C, while even a decrease occured upon herpes simplex virus. The potentiating effect of indomethacin-like drugs was not only observed in bone marrow-derived macrophages but also in mouse peritoneal exudate cells, spleen cells and fibroblasts. Supernatants of bone marrow-derived macrophages (controls, after stimulation by CMA and/or in the presence of the inhibitors) were tested for PGE content by radioimmunoassay and in all cases levels of PGE were at the borderline of detection. Release of PGE was also absent after phagocytosis of zymosan, an established stimulant of PGE synthesis suggesting the capacity to produce PGE being dimished in bone marrow-derived macrophages. Addition of exogenous PGE to CMA-stimulated macrophages in the presence of the inhibitors of cyclooxygenase did not reverse the enhancement of IFN induction by these drugs. Because of these results we conclude that depending on the inducer different regulatory mechanisms of IFN induction in macrophages exist and that other mechanisms than inhibition of PGE synthesis are responsible for the enhancement of CMA-induced IFN induction by indomethacin and similar drugs.
Cancer institute, Chinese Academy of Medical Sciences, Beijing (Peking), PRC
245. Macrophage activation - Association and dissociation of macrophage cytolytic and cytostatic activities D. SUN and Y. ZHANG
The cytolytic and cytostatic effect (on both in vitro growth of tumor cells and IL2dependent lymphoblasts) of mouse thioglycollate-induced peritoneal macrophages treated with TPA or macrophage-activating factor (MAF) was investigated. The results indicated that: 1) TPA at nanogram dose levels induced macrophage cytostatic activity, but it did not make macrophage cytolytic even though IOOO-fold higher doses were
162 . 16th International Leucocyte Culture Conference, Cambridge used; 2) While optimal doses of MAF could activate macrophages to exert both cytolytic and cytostatic effects on tumor cells, suboptimal doses of MAF activated macrophage cytostatic effect only; even lower dose of MAF was enough for activating macrophages to become suppressive to the growth of ILZ-dependent lymphoblasts; 3) Thioglycollate-induced peritoneal macrophages after in vitro culture for 3-5 days lost their responsiveness to MAF to become cytolytic. However, such macrophages were still capable of being activated by MAF to become cytostatic. Thus, the cytolytic and cytostatic activities of activated macrophages could be both associated and dissociated depending on the nature and dosage of the stimulants and the responsiveness of macrophage.
Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, and IUnit of Microbiology and Immunology, Faculty of Health Sciences, The Ben-Gurion University of the Negev, Beer-Sheva, Israel
246. Heterogeneity of macrophage-hybridomas: relation of distinct functions to structure E. TZEHOVAL, S. SEGALl, N. ZINBERG, and M. FELDMAN We report the generation of macrophage hybridomas, obtained by somatic cell fusion between a macrophage-enriched C3H.eB spleen cell population and a drug-resistant MPC-11 myeloma cell line, designated clone 4TOO.1Ll. Screening for hybridomas possessing macrophage properties was carried out by assaying for the presence of two macrophage-specific enzymes: lysozyme and nonspecific esterase. Two hybridomas, E2-7 and E2-10, were selected for further study. We found that a clone derived from E2-7 (E2-7.7) did not express Fc receptors but possessed cell surface Ia molecules. In contrast, clones derived from E2-10 (E210.20 and E2-10.50) possessed Fc receptors but lacked Ia molecules. Opsonized erythrocytes were phagocytosed by E2-10.20 and E2-10.50 but not by E2-7.7. Testing the antigenpresenting capacity of the hybridomas by the response of KLH-primed lymph node cells or enriched T lymphocyte populations to KLH-pulsed hybridoma cells, we found that E2-7.7 caused proliferative responses of both lymph node cells and T lymphocytes, whereas E2-10.20 did not. E2-10.S0 could present antigen only to primed lymph node cells (which are composed of T, Band macrophages) but not to primed T cells. We suggest that E2-10.50 interacts thorough an intermediate accessory cell on antigen-primed T cells. This accessory cell was found to be la-positive and plastic adherent. The difference in antigen-presenting capacity could not be attributed to differences in antigen uptake by the different hybridomas, since all the clones manifested the same level of KLH-pinocytosis. The segregation of functional properties among the hybridoma clones may help clarify the dependence of distinct functions on defined molecular structures.
Harvard Medical School, Boston, MA, U.S.A.
247. Antigen presentation: Its regulation and mechanisms E. R. UNANUE The presentation of many protein antigens to helper T cells requires that the antigenpresenting cells process the protein in intracellular vesicles. The evidence for intracellular
16th International Leucocyte Culture Conference, Cambridge· 163 processing will be reviewed using as antigen the intracellular pathogenic bacteria Listeria monocytogenes and the small protein hen-egg lysozyme (HEL). Both have to be taken up by the macrophage and internalized, after which the immunogenic determinant shuttles to the cell surface. The portion of HEL presented by the macrophage is contained in a 16 amino acid fragment. Macrophages that have processed the antigen can be fixed and are still able to present it to T cells. Antigen presentation by the macrophage is under careful regulation. This regulation is exercised at the level of expression of Ia molecules. We shall review the evidence for the presence of an amplification circuit in which helper T cells recognize antigen on Iapositive macrophages and release gamma-inteferon that, in turn, recruits macrophages to express Ia and amplifies antigen-presenting cell function. The expression of la by macrophages is under negative control in various tissues, in part, modulated by the level of prostaglandins. In the neonate, there is a paucity of la-positive macrophages resulting from PGE2 and alphafetoprotein. Both these molecules are powerful inhibitors of la expression. The inhibition of la in the macrophage may be one factor to consider to explain immunological unresponsiveness and self-tolerance in the neonate.
Max-Planck-Institut fiir Biologie, Abteilung Immungenetik, CorrensstraBe 42, D-7400 Tiibingen, FRG
248. T cells respond to native antigens: no evidence for the necessity of antigen processing P. WALDEN, Z. A. NAGY, and J. KLEIN
In order to investigate the requirements for antigen presentation to antigen specific major histocompatibility complex (MHC) restricted T helper cells, liposomes were produced containing MHC class II molecules and antigen. It was tested whether the antigen fixed to a membrane that contained the appropriate restriction element is sufficient to activate T cells or whether a molecular alteration of the antigen in a processing step is necessary to create the stimulatory structure. In all cases tested such liposomes were capable of stimulating a proliferative T cell response in the absence of antigen presenting cells. These results show that T cells recognize native antigens and that processing is not an essential step in antigen presentation.
Lehrstuhl fiir Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat Bochum, 4630 Bochum, FRG
249. Identification of differentiation antigens on the murine myeloid/ macrophage cell line Ml by monoclonal antibodies (MABS) U. WILLMER, B. BOENKE, and F. W. FALKENBERG The aim of this study is the characterization of differentiation antigens on macrophage populations with mabs. For this purpose, 2 cell fusions were performed in which spleen cells of DA rats immunized with mouse macrophage populations were fused either with X63-Ag8.653
164 . 16th International Leucocyte Culture Conference, Cambridge mouse (MIV: «interspecies hybridomas») or Y3-Ag 1.2.3 rat (MV: «intraspecies hybridomas,,) myeloma cells. The mabs produced by the hybridomas were characterized by indirect immunofluorescence on various natural cell populations and transformed cell lines. As expected, most of the mabs show extensive crossreactions with several cell lines of lymphoid origin. Some of them did, however, exhibit specificity for macrophages alone or for macrophages and one or two other cell types. The Ml myeloblast cell line turned out to be an ideal object to study the sequential expression of differentiation antigens with the help of these mabs. This cell line was derived from a spontaneous myeloid leukemia of a SL mouse. The cells can differentiate spontaneously into mature macrophages. In this differentiation state they become strongly adherent and exhibit macrophage properties. In addition, differentiation can be induced by various agents, such as: ascitic fluid, supernatant from embryonal fibroblasts (L929), LPS and Con A. Mabs MIV 32, MIV 51, MIV 52, MIV 113 and MIV 116 which show crossreactivity with various lymphoid cell types reacted strongly with the non adherent undifferentiated M1 cells but weakly or not at all with the spontaneously and induced (by ascitic fluid or by L929 supernatant) differentiated adherent M1 cells. In contrast, we observed that the mabs which seem to be specific for macrophages, MV 87, MV 114 and M1 70, reacted with spontaneously as well as with induced differentiated adherent Ml cells, but not with the undifferentiated non adherent cells. Mabs MIV 38 and MIV 55 only reacted with spontaneously differentiated M1 cells. Other agents like LPS and Con A did not change the antigen pattern detected by mabs as compared to the original M1 cells. Thus the sequential appearence of differentiation antigens on cells of different differentiation states can be monitored by our mabs. The antigens recognized are currently being further characterized.
Department of Microbiology and Immunology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan
250. Regulation of immune response by pre administration of cells briefly pulsed with antigen in vitro. III. Induction of antigen specific suppressor cells by UV treated antigen pulsedJ774 cells K. YOKOMURO, A. MABUCHI, O. TSUCHIYA, and Y. KIMURA
The intravenous administration of syngeneic spleen cells (SPCs) briefly pulsed with antigen (Ag) in vitro, results in a profound state of IgE antibody unresponsiveness. This suppression is mediated by two different mechanisms: one of them is responsible for the immediate tolerance which is induced most effectively by antigen pulsed T cells in the subpopulation of SPCs (KOJIMA et ai., 1984) and the other is responsible for the suppression transferred by suppressor cells (YOKOMURO et ai., 1983). To investigate the first step of suppressor cell induction, one of macrophage like cell lines, J774 cells which originated from BALB/c and could not present Ag in vitro to syngeneic primed T cells were pulsed in vitro with DNP-KLH or beef insulin and injected to BALB/c mice (1st recipients). Six days after, SPCs of 1st recipients were transferred to syngeneic mice (2nd recipients). Two-4 hrs later, 2nd recipients were immunized with Ag plus alum. Their IgE levels were measured with passive cutaneous anaphylaxis (PCA) in rats. Following results were obtained: (1) The induction of Ag specific suppressor cells were dependent on the dose of injected Ag pulsed ]774 cells. (2) Ag pulsed ]774 cells exposed to UV light were capable of inducing the immediate tolerance but not capable of inducing the suppressor cells. (3) In some experiments 10 days after the injection of Ag pulsed SPCs, 1st recipients were immunized with Ag plus alum. Four days later, SPCs from 1st recipients were
16th International Leucocyte Culture Conference, Cambridge· 165 transferred to 2nd recipients and then they were immunized. By the immunization before SPCs transfer, Ag pulsed ]774 cells exposed to UV light also could induce suppressor cells. These results suggest that two signals may be necessary for inducing suppressor cells. First one is antigen itself pulsed on UV treated ]774 cells and second one is factors/cells activated by immunization following the transfer of UV treated Ag pulsed cells. KOJIMA, N., A. MABUCHI, K. YOKOMURO, and Y. KIMURA. (1984). Suppression of IgE antibody response by preadministration of antigen-pulsed spleen cells II. char,cteristics of immediate tolerance induction. Int. Archs Allergy app!. Immun., in press. YOKOMURO, K., A. MABUCHI, M. SAIZAWA, N. KOJIMA, A. S. ROSENTHAL, and Y. KIMURA. (1983). Regulation of immune response by preadministration of cells briefly pulsed with antigen in vitro I. suppression of IgE antibody response by antigen pulsed spleen cells. Int. Archs Allergy app!. Immun. 69: 98.