Workshop 4 Lymphokines - Cytokines

German Cancer Research Center, Institute for Immunology and Genetics, 1m Neuenheimer Feld 280, 6900 Heidelberg, PRG

1. Does the differentiation of cytotoxic T lymphocytes require IFN-y?

L. DAUM, W. FALK, W. DROGE, and D. N. MANNEL

In order to study the role of IFN-y for the induction of cytotoxic T lymphocyte (CTL) responses, microculture systems were established that were entirely dependent on addition of helper factors. In Sd-cultures comprising 1 r:f thymic responder cells and 3 X 105 x-irradiated (lS00R) TNP-modified syngeneic stimulator cells endogenously produced IFN-y was meas­ured during generation of cytotoxic responses with the help provided by semipurified IL2~ prt!parations devoid of IFN-y; the maximum of this endogenous production of IFN-y was on day 3. For the induction of cytotoxic responses in cultures of 105 thymic responder cells and 3 X 105 glutaraldehyde-fixed TNP-modified syngeneic stimulator cells all helper mediators necessary for the induction of CTL function were provided by Con A induced spleen cell supernatants. Besides IL2 an early acting T cell cytotoxicity inducing factor 1 (TCFl) and a pH2labile late acting helper factor (TCF2) were required for the generation of CTL (1). In addition, these supernatants also contained IFN-y which is also pH 2 sensitive. However, the loss of the helper potential of Con A supernatants after treatment at pH 2 was reconstituted only by TCF2 preparations but not by IFN-y. Addition of various IFN-y preparations or various anti IFN-y antisera did not show any eHect on the induction of cytotoxicity. These data suggest that exogeneously added IFN-y does not influence the induction of CTL from thymic precursor cells. One can also conclude that even if IFN-y was essential the necessary amounts were endogenously produced and not accessible to neutrali:£ation by antibodies to IFN-y:

1. W. FALK, D. N. MANNEL, and W. DROGE. 1983.]. Immunol. 130: 2214.

forschungsinstitut Borstel, Parkallee 1-40, 2061 Borstel, FRG

2. Binding of interleukin 2 to target cells: Expression of two classes of binding sites on stimulated T-lymphocytes

H.-J. HORST, G. GRINGEL, and H.-D. FLAD

The biological action of interleukin 2 (IL2) is obviously dependent on its binding to nructurcs (n:u,::pwfS) on the surface of IL2-responsive cells (target cells). Recently we developed a binding assay which allows to discriminate between high affinity and low affinity binding sites. Target cells were incubated with an excess of highly purified human IL2

Workshop 4: Lymphokines - Cytokines 31

prepared from mononuclear cells by RP-HPLC. Unbound IL2 was separated from cell bound I L2 by sedimentation through fetal calf serum. IL2 bound with low affinity was removed by a differential dissociation step. Total bound, or high affinity hound IL2, respectively, was measured by a lH -thymidine incorporation test after di sruption of the target cells. In T­Iymphoblasts, most of the bound IL2 was found in the low affinity fraction which showed a di ssociation half time of less than 1 min. Only 15 % was bound with high affinity with a dissociation half lime of 80 min . Both types of binding sites were heat labile at 56 °C and sensitive La trypsin (3 min at 37°q indicating the protein nature of the binding stru cture. T he kinetics of receptor expression in mitogen or antigen stimulated target cells of different origin will be described. It is concluded that stimulation of III target ce lls induces 2 classes of binding sites which have different affinities but which both are specific for lL2. It remains to

be investigated whether or not the two classes each have a distinct physiological role in the receptor mediated transfer mechanism of the JI.2-specific biochemica l signal.

Universitatsklinik fur Jnnere Medizin, lnnsbruck, Austria

3. Interferon gamma enhances alloreactivity by induction of expression of class II antigens

Ch. HUBER, J. TROPPMAIR, G. GASTL, D. NlEDERWIESER, and R. MARGREITER

Whereas it is generally accepted that IL-2 enhances alloreactivity by virtue of its capacity to maintain growth of activated T cells only little is known about the role interferon-gamma (IFN-gamma) might play in regulating alloreactivity. \VIe report on three independent findings suggesting that IFN-gamma also enhances al lorcactivity but that tbis effect is mediated by induction of HLA-DR antigen expression: 1) preincubation of pnMNC with IFN-gamma increases both the numbers of HLA-DR + cells and the capacity to stimulate allogeneic T cells, 2) monoclonal antibodies directed against IfN-gamma reduce both cellular growth as well as the generation of cytotoxic T cells in MLCs, when PBMNC cells were used as stimulators, 3) when allogeneic lymphoblastoid lines were used as stimulators which invariably express a high density of HLA-DR on their surfaces, no such blocking effect was observed. We thus conclude that IFN-gamma released by activated T cells in response to allogeneic HLA-DR determinants induces further expression of these antigens on previously ncguive stimulator cells which than in turn induce otber T cells to further release lymphokines. It thus appears that Iymphokines may enhance alloreactivity by twO different means: by modifying expression of class Il antigens on foreign tissues and by maintaining the growth of T cells activated by these antigens.

Dept. of Dermatology, Univ . of Freihurg, FRG 1 D ept. of D ermatology, Univ. of Vienna, Austria 1 D ept. of Clinical Immunology, Univ. of nero, Switzerland

4. F urther character ization of adherent cell derived granulocyte activating mediators (GRAM)

A. KApP, T. A. LUGERl, H. W OKALEK, and B. STADLER2

We recencly reported that human adherent ceUs stimulated with LPS produced gran ulocyte activating mediato rs (GRA.J.Vl). These GRAM induce a long-lasting activation of human

32 XVI. Meeting of the Society of Immunology

polymorphonuclear leukocytes (PMN) as measured by Lucigenin-dependent chemilumin­escence (Cl). Enhancement of O2 -production could also be detected using cytochrome-C reduction. GRAM activity in the supernatants (SN) was generated after stimulation with PHA, silica particles, and lPS. LPS at concentrations of 50 to 500 ng/mt was the most effective stimulus. The generation was time-dependent and appeared to be maximal at 24 h. Irradiation with 2000 R did not reduce GRAM production, whereas addition of protein synthesis inhibitors resultt!d in significantly decreased release of GRAM. Furthermore GRAM was heat labile and sensitive to trypsin treatment. Extensive dialysis against saline diminished me activity. Since partially purified interleukin-l (IL-l) exhibited GRAM activity, we investigated whether this activity was associated with IL-l or other copurifying lymphokines. Recombi­nant human Interferon 112 and y failed to induce GRAM generation. SN of LPS stimulated adherent cells were fractionated using HPLC gclfiltration. Subsequently GRAM - and lL-1 -activity were tested in parallel. Two peaks of GRAM activity were recovered which were different from IL-1 activity. Since IL-1 apparently was not responsible for GRAM-activity, our data indicate that adherent cells upon stimulation may release an additional immune­modulating factor, which regulates granulocyte activity.

Dept. of Dermatology, Univ. of Freiburg, lGerman Cancer Research Center, Heidelberg, 2Thomae GmbH, Biberach a. d. R., FRG

5. Modulation of granulocyte oxidative response by recombinant interferon u, and y as measured by lucigenin-dependent chemiluminescence

A. KAPP, H. KTRCHNER1, H. WOKALEK, and W. BERTHOLD2

We were recently able to demonstrate that a stimulation of human adherent cells by LPS results in the generation of mediators activating human polymorphonuclear leukocytes (PMN) to produce reactive oxygen species (ROS). Our interest was to investigate whether these mediators may have some connection with interferon (IFN). In order to exclude the possible influence of impurities in IFN preparations, only recombinant human IFN U2 or IFN y of a specific activity of 3 X 108 IU/mg protein for IFN U2 and chromatographically pure IFN y (1-2 x 107 laboratory Units/mg protein) were used. Lucigenin-dependent chemiluminescence (CL) of PMNs was measured to assess the production of ROS. IFN Y only at a concentration of more than 10 ng/ml could elicit a minimal CL response in PMNs. When PMNs were incubated with IPN y for 1 h and then stimulated with chemotactic peptide f-met-phe (FMP, 2 x 10- i M), zymosan-activated serum (ZAS). Zymosan particles, or phorbol-myristue­acetate (PMA, 100 nglml), the Cl response was significantly increased. IFN U2 had no such effect at any concentration tested. A short pretreatment of 5 min with IFN y decreased the ZAS response, but did not affect the reaction to the other stimuli. The possibility of a generation of IPN by PMNs during the assay could be excluded as no IFN activity could be detected in an antiviral assay after stimulation of PMN for 6 h with PolyI X PolyC, LPS, Con A, C. parvum, PMA, Zymosan or FMP. The modulation of granulocyte reactivity by IPN Y may be important in the regulation of the anti-inflammatory response of PMN.

Workshop 4: Lymphokines - Cytokines 33

Immunopharmacology, Medical School of Hannover, and German Cancer Research Center, Heidelberg, FRG

6. Induction of tumor cytotoxicity in macrophages by macrophage activating factors (MAFs): Role of interferon y (IFN-y) and macrophage cytotoxicity inducing factor (MCIF)

B. KaZAN, D. LOVEIT, C. KUBELKA, P. H. KRAMMER, and D. G EMSA

Recently it has been shown that IFN-y displays MAF activity if lipopolysaccharide (LPS) is present as an additional triggering signal. These findings, however, do not exclude the existence of other MAF systems thar may be equally efficient in activating macrophages to tumor cytotoxicity. We tested this possibility by examining the activation patterns of macrophages from LPS-high responder DBA!2 and LPS-low responder C3H/HeJ mice. Resident peritoneal macrophages were either activated by ConA-induced supernatants of the T cell elones 96 and 29, which contain both IFN·y and MAF (MAf96 and MAF29), or by recombinant IFN-y plus LPS. Bmb DBAI2 and C3H/ HeJ macrophages cou ld be activated by MAF96 or MAF29 to cytotoxicity against tumor cells and schistosomula. In COntrast, only DBAI2 but not C3H/H eJ macrophages were activated by recombinant IFN-y plus LPS. C3 HI He] macrophages could be efficiently primed b y recombinant IFN-y, howtvtr, they required for induction to tumor cytotoxicity low concentrations of MAF96 or MAF29 which alone were entirely ineffective in activating macrophages. Thus, MAF96 and MAF29 contain, besides IFN-y, another Iymphokine capable of fully activating C3H1HeJ macrophages to tumor cytotoxicity. The biochemica1 properties of this Iymphokine, tentatively named MC IT-, remain unclear. It is known that this lymphokine is less sensitive to heat and low pH treatment than lFN-y. These data point to the existence of a MAP system other than IFN-y plus LPS, which may become operative in activating macrophages, particularly under those circum­stances in which LPS is not availab le as a triggering signal.

Hnd Department of Dermatology, University of Vienna, Vienna, Austria and Institute of Clinical Immunology, University of Bern, Bern, Swi t1.erland

7. Production of a monoclonal antibody directed against human interleukin 1

A. K()CK, M. D ANNt:R, B. M. STADLER, and T. A. LUGER

H uman Interleukin 1 (IL 1 ) was generated under serum free cond itions by stimulating adherent cells with Silica (50 flg/ml). IL 1 containing supernatants were lyophilized, desalted using Bio Gel P-6 DG desalting gels and partially purified using HPlC. Bio Gel TSK 125 size exclusion chromatography. This partially purifitd material was used to immunize BALB/c mice. After immunization spleen cells were hybridized with BALB/c myeloma SP 3 cells. Supernatan ts of the hyb ridoma cuhurts were screened for their capacity to inhibit IL 1 mediated enhancement of mitogen induced C3H/He thymocyte proliferation (> SO %) and to bind to partially purified II. t using a dot-immunobinding assay. After expansion and cloning one cdl line was sdecttd for ascitic antibody production. Ascitic anti IL 1 antibody (n IL 1) was of IgG Immunoglobulin class and bound to highly purified human IL 1 even when diluted t :2500. In addition n IL 1 inhibited > 80 % of IL 1 mediated enhancement of thymocyte

34 XVI. Meeting of the Society of Immunology

proliferation, neutralized > 90 % of IL 1 dependent fibroblast proliferation and blocked > 80 % of the IL 1 mediated IL 2 production by llUman T lymphocytes in culture. a IL 1 has been shown to bind to human ETAF (epidermal cell tbymocyte activating facwr) and to block the biological effects of this keratinocytc derived cytokine which appears w be identical to IL 1. Furthermore a IL 1 not only blocked the biological effects of human IL 1 and ETAF but also neutralized murine IL 1 as well as murine ETAF activity.

In contrast u IL 1 did not inhibit the biological activities of Interleukin 2 (IL 2) or Interleukin 3 (IL3) and did not bind to recombinant IL2 in a dot i.mmunobinding assay. Furthermore the proliferative activities of a macrophage (U 937), T cell (HSB2) or B cell (will 2) lines were not affected by the presence of IL 1. A single passage of IL 1 containing supernatant over a a IL 1 coupled CNBr sepharose column removed> 90 % of the IL 1 activity present. Elution of IL 1 activity from affinity columns yielded> 20 % of bound IL 1 activity. These data provide first evidence of a monoclonal antibody directed against IL 1. which may be helpful for the development of new detection assays for IL 1 and for further isolation. purification and characterization of this cytokine.

Medical School Hannover and lnst. for Immunol.. Heidelberg, FRG

8. Activation of glomerular mesangial cells (MC) by terminal complement components: Stimulation of prostanoid and inter­leu kin 1 (IL-l )-like factor release

D. LOVEIT, G. HANSCH, K. RESCH, D. GEMSA

Complement deposition in the glomerular mesangium is a histologic hallmark of many forms of immune-mediated glomerulonephritis (GN). While prior work has emphasized the role of immune complexes with bound complement, it has become recently evident that the terminal membrane attack complex (MAC) of C5-9 alone may be found in significant quantities in the mesangium. It is not clear from these histologic studies whether C5-9 deposition is an epiphenomenon, or represents a further mechanism for the initiation of glomerular injury. We therefore assessed the ability of C5-9, in nonlytic concentration, to

stimulate from cultured rat MC the release of prostanoids (pGE2• prostacyclin-PGI2, and thromboxane-TXB2) and a MC-derived IL-I-like factor, which exhibits MC autostimulatory growth activity.

PG release after 6 hrs exp: (N = 6, trip!' determ.; ngl100 lAg cell prot.)

PGE2 PGI, TIm,

Medium 1.48 ± 0.2 <0.35 <.009 Pre-incub. C5-9 1.13±0.1 <0.35 <.009 C5-9 32.80 ± 0.9 11.7 ± 0.6 0.60 ± .04 Ca ionophore 16.50 ± 1.5 62.5 ± 3.5 1.01 ± .03

IL-l-like factor release was stimulated> 30-fold after 6 hrs. stimulation (Contr: 63 ± 4 U I 100 flg protj C5-9: 1983 ± 1800 UI IOO I-lg prot). These findings indicate that cultured renal mesangial cells are highly responsive to C5-9 complex, and suggest that deposition of this complex in the mesangium during the course of immune-mediated GN may playa major role in the hemodynamic and cellular proliferative changes found in these disorders.

Workshop 4: Lymphokines - Cytokines . 35

Institute for Applied and Experimental Oncology and lInd Department of Dermatology, Uoiv. of Vienna, Vienna, Austria

9. Human epidermal cells and squamous carcinoma cells synthesize a cytokine which augments natural killer cell activity

M. M ICKSCHE, M. COLOT, A. UCHIDA, A. KOCK, and T. A. LUGER

Nonnal as well as transformed epidermal cells ( EC) recently have been reported t.o produce a cytokine - epidermal cell derived thymocyte activating factor (ETAf-) - which according to its b iological as well as biochemical properties is indistinguishable from macrophage derived Interleukin 1 (IL l). Furthermore, according to previous experiments partially purified ETAF but not IL 1 has been demonstrated to augment human natural killer cell (NK) activity. In me present study the effect of supernatams derived from normal EC and a h uman squamous carcinoma cdl (SeC) line were tes ted fo r their effects on NK cell activity. EC as well as SCC derived supernatants were able to augment in vitro N K cell activity of blood lymphocytes against K 562 cells. In contrast, adherem cell derived, IL 1 containing supernatants again did not affect N K cell activity . Furthermore, incubation of enriched large granular lymphocytes (I.GL)-isolated by discontinuous Percoll gradient centrifugation and depletion of high affi nity sheep erythrocyte rosetting cells-with EC as well as SCC supernatants resulted in a significant augmentation of NK cell activity whereas LGL-depleted fractions wt!re not active. For biochemical characterization SCC cell dt!rived supernatants were concentrated 100 X by Amicon YM 10 ultrafiltration and subsequently subjected to high pressure liquid c hromato­graphy (HPLC) gel filtration using a Bio Gel TSK 125 column. ETAF and the NK cell activity augmenting facto r (ENKAF) exhibited a similar molecular weight between 30 Kd and 10 K d. Using reverse phase (RP) chromatography a dear separation of ETAF and ENKAF could be achieved, w hereas most of the ENKAF was found to elute at low concentrations of acetonitrile within 4 d istinct peaks, ETAF activity eluted as 5 peaks within the higher concentration of the gradient, In addition minute amounts of Interferon (lFN) also present in the see supernatants when chromatographed upon RP dearly ',Vere separated from ENKAF. Furthermore ENKAF does not comain Imerleukin 2 (IL 2) activity. These results indicate that normal as well as transformed EC release a unique cytokine - ENKAF - whieh augments N K cell activity of LGL and is distinct from ETAF, IL 2 and lFN.

Deutsches Krebsforschungszemrum, Institut fur Immunologie und Genetik, 1m Neuenheimer Feld 280, 6900 H eidelberg, FRG

10. T cell cytotoxicity inducing factor 1 (TCF 1) is a product of activated T cells

E. ORBACII-YURUKA, D. N. MANNEL, W . D ROGE, and W. FALK

Generation of cytotoxic responses in cultures of 105 thymocytes and 3 X 105 glutaraldehyde­fixed TNP-modified spleen stimulator cells is dependent on at least three different activities called TeFl, TeF2 and IL2 ( I), Te Fl activity was tested i n presence of excess amounts of IL2 and TCF2 which by itself did not induce cytotoxicity. TCFI was found in supernatants of

36 . XVI. Meeting of the Society of Immunology

ConA stimulated spleen cells, eonA pulsed spleen cells and mixed lymphocyte cultures. More homogeneous cell populations as sources for TeFl were investigated i n order t o determi ne whether TCFl is a p rod uct of macrophages or T cells. TCFI was found in supernatants of stimulated EL4 thymoma cells and ConA pulsed T cell clones but JlUL in supernatants of stimulated P388Dt or PU5.18 macrophage-like cell lines. Funher evidence for production of TeFt by T cells was provided using another TeF l-limited microculture system. When I O~ to 2 X lOs thymic responders were cultured with varying numbers of allogeneic thymic (X­irradiated) stimulator cells and saturating amounts of IL2 and TCF2 we found that the system was limited f o r endogenously produced TeFl . No cytotoxic response was obtained when the stimulator ceUs were absent, syngeneic or UV-irradiated indicating that the IL2 preparation by itself did not induce in thymocytes production of sufficient amounts of TeFl. The cytotoxic response observed after 5 days of culture was dependent on the dose of allogeneic metaboli­cally active thymic stimulator cells and was H -2 specific. 'raken these data together one can conclude that TeFt i s a product of mitogenically or antigcnically activated T cells although a possib le p roduction also by contaminating macrophages cannot be ruled out.

\. W. FAL., D . N. MANNEL, and W. DROGE. 1983. J. Immunol. 130: 2214.

Department of Medicine, D ivision of H aernawlogy and Oncology, University of Marburg, FRG

11. Purification and characterization of a colony-stimulating factor (CSF) produced by a ce11line from a human carcinoma of the urinary bladder (UBC)

K. -H . PFLDGER, H. KAPMEYER., D. STACI r, W. -D. CASSEL, K. HAVEMANN

The production of mature granulocytes and monocytes from haemopoictic progenitor cells is dependent on the pre.<;ence of a class of glycoproteins which have been termed colony­stim ulating factors (CSFs). In addition functional activation of granulocytes and monocytes are achieved b y these factors. The group of human CSF!> is quite heterogenous in terms of biochemical and biological properties. The factors are produced by different cells (i.e. mature monocytes. stimulated lymphocytes and spleen cells, placenta cells and embryonal tissues) and ectopic secretion by several tumors is well known.

Of 55 rumor cell lines established i n o ur laboratory the supernatants of 6 showed considerable colony-stimulating activity measured by bioassay using a rwo-Iayer technique and mononuclear cells of human bone marrow. The csr: of one of these lines originating from a human adeno carcinoma of the urinary bladder is purified and chancterized. The character­ization of the cell line will be presented.

'The purification of the CSF I!> achieved according the fo llowing scheme: a) Supernatants of serum free cel l culture are concentrated and desalted, b) Sephadex G 100 chromatography, c) HPLC o n a C8-wide pore column in reversed phase mode. Alternatively to c) we used FPLC anion exchan ge chromatography followed by a HPLC geJfilrration step.

The CSF from the USC-cell lines shows the following biochemical and biological proper­ties : a) H eterogenei ty in tcrms of molecular weight and isoelectric point, b) Stability up to

56 °C, by addition of serum albumin higher temperatures arc tolerated, c) pH-stability from pH 3-11, d) Resistant biological activity againsl treatment with neuraminidase, e) Production of mixed granulocyte and macrophages containing colonies without eosinophils, f) No activity in the mouse system.

Workshop 4: Lymphokines - Cytokines 37

I Institut fur Immunologie dee Universitat, 6500 Maim., 2 Abteilung rmmunologie der Univer­sitar, 7900 Ulm, 31mmunologische Forschungseinheit, Klinikum Steglitz, Feeie Universi tat Berlin, West Germany

12. Analysis of intcrlcukin-2 receptor expression on an antigen­dependent T cell line with anti-receptor antibodies

A, B, RESKE-KUNZ1, D. v. STELDERN2, E. Rom:i, H. OSAWA\ and T. D lAMANTSTElN3

We studied the basis for the antigen-dependence of long-rerm proliferation of insulin­reactive T cell lines, which lose growth potential in imerleukin-2 (IL-2) containing medium and require renewed antigenic challenge to regai n responsiveness towards rL-2. The presence of IL-2 receptors on the surface of these cells was monitored at various time points after antigenic stimulation utilizing monoclonal antibody AMT-13, which detects murine IL-2 receptors. Evidence was obtained, that blasts, cultured in IL-Z containing medium for prolonged periods of time, have undetectable levels of IL-Z receptors on their cell surface. Following specific antigenic stimulation IL-Z receptors arc expressed in high density. The expression of IL-Z receptors is transitory, being maximal on day 2 after antigen challenge and having declined to low levels by day 13. The decreased display of IL-2 receptors observed On day 13 was not due to a poor cell viabili.ty, since these cells could be induced by insulin and splenocytes to multiply extensively. Data on the proliferative capacity of T blasts, cultured in IL-Z containing medium in the presence of anti-I-A antibodies suggest that the transitory expression of IL-2 receptors does not depend on permanent antigenic stimulation .

Supponed by the Deutsche Forschungsgemeinschaft, SFB 107 (Mainz),

Max-Planck-Institut fur Immunbiologie, Freiburg, FRG

13. Regulation of the generation of cytolytic T lymphocytes by soluble mediators derived from cloned T killer cells

M. M. SIMON and H EIDRUN MOLL

We have previously shown that cloned H-Y specific cytotoxic T cells (CTLL) can elicit both suppressive and helper activities on the development of cytolytic T lymphocytes (CTL) from their precursor cells (CTL-P) in vitro (M. M. Simon et al., Cellular ImmunoL, in press) . Here we show that H-Y specific and H-2 D b restri cted, Lyt-I -2+ C57BLl6 CTLL, when stimulated with Con A, secrete soluble mediators that interfere with the maturation of CTL by inhibition of the activation and/or expansion phase of CiL-P. The suppressive activity observed is antigen independem but restricted to the H_Zb haplotype, It was also found that one of the targets of the CTLL derived factor is the Lyt-Z- population and that the suppressive activity acting on the generation of CIL is noc reversed by the addition of exogenous sources of T cell growth factor (EL-4-supernatant). In addition the data demonstrate that the inhibitory activity fOUJld in mitogen induced eTLL supernatant does not correlate with the content of interferon-yo These results indicate that cytotoxic effector cells are able to regulate the differentiation of their precursor cells by virtue of a soluble mediator(s) which may block the maturation of CTL at different stages of their development .

38 . XVI. Meeting of the Society of Immunology

Institut fur Immunologie der Universitat Maim., FRG

14. Production of synergistic growth activity(ies) (SGA) by T cell hybridomas

E. SPAETH and E. RODE

Interleukin-2 (JL-2) has been shown to act 3.<; a growth factor for helper as well as killer T cell clones. Nevertheless, such clones need feeder cells in addition to IL-2 for growth at limiting cell numbers indicating additional growth requirements. Moreover) Con A-activated spleen cells do not respond to IL-2 alone at limiting cell or Con A concentrations or when exposed to a preactivating Con A pulse of limiting duration again indicating the need for additional activation and/or growth signals. We could demonstrate that the supernatant of the T cell line ST2/G contains a growth promoting activity (SGA) which acts synergistically with IL-2 on T cells cultivated under limiting conditions. In order to dissociate the SGA from other lymphokines produced by this T cell line and to obtain stable, fast growing SGA-producing cells, the ST2/G-line was fused with BW 5147 thymoma cells. T cell hybridomas could be isolated and selected that are producing SGA while other Iymphokines are found in the supernatants at lower levels than in those of the original ST2/G- line. The properties of these clones are discussed.

1 Department of Dermatology I., 2Department of Dermatology II., University of Vienna, Austria

1 Department of Biochemistry, Thomae GmbH, Biberach, PRG

15. y-Interferon stimulates HLA-DR biosynthesis of cultured human keratinocytes

BEATRIX VOLe-PLATZER l, T. LUGER1, H. LEIBL l, GABRIELE ZAH~, G. STINCLl

Under certain pathological conditions, human keratinocytcs synthesize and express HLA­DR antigens. Assuming that soluble mediators might be responsible for this phenomenon, differentiating, primarily DR - keratinocytes were grown in the presence or absence of mixed leukocyte culture supernatants and tested for Ia antigen expression. After 6 days of culture, keratinocytes displayed surface-bound HLA-DR a/~ complexes when exposed to mixed leukocyte culture supernatants but not when cultured in media alone. These HLA-DR moieties on keratinocytes result from active biosynthesis as evidenced by the demonstration of the intracytoplasmic HLA-DR y (invariant) chain within these cells. in view of reports that interferon-y promotes la-production in a variety of cell types, we reasoned mat this Iym­phokine might be responsible for the la-inducing property of mixed lymphocyte culture supernatants. Indeed, recombinant interferon-y, but not interferon-a proved to be a potent stimulator of Ia expression by keratinocytes. The further finding that this event can be prevented by the addition of a monoclonal anti-interferon-y-antibody strongly suggests that this cytokine is directly responsible for !-ILA-DR production by keratinocytes. The interfe­roo-y induced acquisition of HLA-DR antigens by primarily DR- keratinocytes may provide a useful tool to study the role of these alloantigens in T cell activation and may also add to our understanding of mechanisms operative in bidirectional epidermal cell - T cell interactions.