t lymphocytes: effector functions

14th International Leucocyte Culture Conference, Heidelberg 143

T Lymphocytes: Effector Functions

Department of Cell Biology, The Weizmann Institute of Science, RehovO[, Israel, and Molecular Biology Institute, University of California, Los Angeles, California, USA

The mechanism of T lymphocyte-mediated lysis

GIDEON BERK E and WILLIAM R. CLAH

CytOtoxic T lymphocytes (CTL) are probable effectors in allograft rejecrion, antivirus responses and possibly in tumor immunity. Yet, the precise mechanism whereby CTL interact with and lyse target cells (TC) has remained unknown. The sequence of events associated with cytolysis has bcen resolved and grouped into three stages: binding of the CTL and TC; programming for lysis; and TC dissolution. Although these ~tudies suppOrt the notion that TC binding and delivery of the lethal hit [Q the TC are distinct steps in the lytic process. they by no means demand the involvement of moleculary distinct entities of the CTL in the binding and lytic steps. Ncw data obtained in our laboratorie.~ have Icd us to the conclusion that CTL arc not equipped with a distinct cytolytic entity that is activated upon contact with TC. Rather, we would Jike to propose that under permissive conditions TC destruction follows as a natural and inevitable consequence of CTL-TC binding. In the single C1'L entity model we are proposing. lysis occurs as a direct consequence of binding of the CTL MHC receptor to TC MHC antigens. Our underlying assumption is that the integrity of the plasma membrane as a permeability barrier depends on appropriate interactions of the constituent lipids with each other and with integral membrane proteins, particularly transmembrane proteins. We assume that TC membrane lipids and the transmembrane MHC proteins coexist in the lowest energy state; interference with their freedom to interact properly creates a thermodynamically unstable interface between them, resulting in loss of the permeability barrier function of the membrane. Experiments have shown that CTL-TC binding, through specific CTI.. surface receptors and TC MHC components, occurs across an extensive porrion of the twodimensional surface of the CTL and TC. We propose that physical restriction, rearrangement, or conformational changes of the MHC proteins in the plane of the TC plasma membrane, by the imposition of a two-dimensional crosslin king grid consisting of the CTL surface receptor (non-transmembrane), creates instabilities at the MHC protein bilayer-lipid interface. These distortions in the hydrophobic regions of the MHC proteins result in molecular mismatching at the protein-lipid interface, which in turn lead to inc reased membrane permeability and 'fC di .~solution. Our theory that a single CTL entity mediates both binding of TC MHC components and TC lysis is not in conflict with the lectin (Con A)-dependent CTL-mediated lysis since in this nonspecific system too we have shown that TC MHC antigens are required for lysis to take place.

Service de Nephrologie, H opital Paul Brousse, Universite Paris-Sud, 94800 Ville)uif, Francc

In vitro responses of anti-idiotypic autoimmunisation in the rat

B. CHARPENTlER, PI!. LANG, B. MARTIN, and D. FRlr.S

It is now generally accepted that clones of circulating T Iymphocyte.~ bear spec ific receptors for alloimmune determinants. It is obvious that specific elimination of recipient clone against

144 . 14th International Leucocyte Culture Conference, Heidelberg

potential donor would have enormous significance in organ-transplantation. This depletion of dones could be mediated by anti-receptor antibodies or by an auto-cytotoxicity phenomenon and directed against cells bearing such sophisticated specific receptors. We investigated the effect of autoimmunisation with mixed-lymphocyte culture (MLC) generated cells upon in vitro proliferative and cytotoxic responses using inbred strains of rats (L. DA, BN) and their Ft hybrids (LBN, LDA, BNDA). Specific clonal expansion of cells bearing receptors for a given set of alJoantigcns were generated in a macro-MLC using P against Ft cells. After 5 days of culture, cells were harvested and separated according to their size by 1 g velocity sedimentation and reinjected to animal of the responding strain. Pure separated blast cells (> 96 %) were injected IP (107 cells) with complete Freund's adjuvant, and incomplete Freund's adjuvant was used for the boosters. After 3 IP injections at 2~week intervals, autoimmunized rats disclosed at various degrees evidence for classical anti-idiotypic antibody with/without loss of specific alloreactivity. In the group of unresponsive rats a secondary "in vitro .. restimulation did not give rise to cytotoxic T lymphocytes. The cytotoxic capacities of spleen cells of such autoimmunized rats exhibited several patterns: 1. cytotoxicity directed against the specific alloreceptor relevant to the stimulating strain and accounting for the immunological unresponsiveness. 2. cytotoxicity directed against the allodeterminam. 3. when autoimmunization was prolonged over 5 injections, a cytotoxicity against self-determinant appeared. Thus, autoimmunization with idiotypic receptors would induce a state of immune dysregulation where rat T lymphocytes can display aggressive patterns of anti-receptors reactivity, alloreactivity and self-reactivity which may have great significance for the understanding of the immunological network.

INSERM U25, Hopital Necker, Paris, France, Service de Diabetologie, Hopital Bichat, Paris, France

Lymphocytes and sera from diabetic patients suppress insulin release in vitro

M. DEBRAy-SACHS, C. BOJARD, P. SAl. R. ASSAN

Lymphocytes from diabetic and control subjects were incubated in vitro with dispersed DBAn mouse Langer-hans islet cells. Insulin and glucagon secretions were stimulated by glucose and arginine respectively. Insulin release was inhibited by lymphocytes from 21 OUt of 22 insulin dependent diabetic (IDD) patients with associated autoimmune diseases and by lymphocytes from 5 out of 8 recent onset IDD patients. Insulin release was not modified by lymphocytes fmm 30 control subjects, 7 non insulin dependent (NIDD) patients and 5 non diabetic patients with autoimmune diseases. The arginine-stimulated glucagon release was maintained when tested in all 4 groups, Similarly, when the corresponding sera were icubated with Langerhans islets in the presence of complement, insulin re1ca.~e was inhibited by sera from IDD patients with associated autoimmune diseases (11 out of 17 tested) and 1 recent onset TOD patient (OUt of 5 tested). Sera from NIDD patients and non diabetic subjects with or without autoimmune diseases did not suppress insulin release. Glucagon release was maintained when tested. Immunoglobulin G (IgG) fractionated from 9 of the IDD sera inhibited insulin release. while globulin-free sera did not. Fractionation of IgG and the use of human Langerhans islets increased the number of positive IDD sera. All the positive sera were also positive for islet cell antibodies as detected by immunofluorescence technique (but not the converse). Lymphocytes from all patients with cytotoxic sera also suppressed insulin release from mouse dispersed islet cells in vitro. Thus, lymphocytes and IgG in the presence of complement actually suppressed insulin release in vitro. It was efficient on normal human


islets. selectively directed against beu cells, and detectable, in patients with autoimmune diseases, only when diabetes was present. These data are consistent with an actual contribution of humoral and cellular anti-islet immunity to the patho-physiology of some forms of insulindependent diabetes.

Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France

Monoclonal antibodies as probes to study the mechanism of T cellmediated cytolysis

P. GOLSTf::1N and M. PIERKES

To gain a better undemanding of the mechanism of T cell-mediated cytolysis at the molecular level, B cell hybridomas making monoclonal antibodies directed against structures involved in T cell-mediated cytolysis have been derived, by fusion of rat spleen cells sensitiz.ed against mouse cytolytic T cell populations and Y3 Ag 1.2.3 rat myeloma cells. They were selected on functional rather than structural grounds for their ability to inhibit a cytolytic reaction. Four such inhibitory antibodies have been studied to-date. One of them, called H35-89.9, was inve.~tigated in more detail. It inhibited T cell-mediated cytolysis, at concentrations down to the nanomolar range, even in the presence of Concanavalin A (Can A), most probably at the lethal hit stage of cytolysis. It also reversibly inhibited antigen-induced and Can A-induced T cell proliferation, but not LPS-induced B cell proliferation, at a stage subsequent to the production of T cell-growth factor. H35-89.9 antibody acted on effector or responder cells, rather than on target or presenting cells. Further details will be given on the corresponding cell surface antigen, the molecular weight and tissular distribution of which are different from that of Lyt-2 and of the molecules recognized by our other inhibitory antibodies. These results show that there is at the surface of cytolytic T cells more than one molecule. the recognition of which by an antibody leads to inhibition of cytolysis. They also suggest that H35-89,9 antibody inhibits a step common to lethal hit and to induction of proliferation, which may be related to lymphocyte activation. Further results on these and other inhibitory antibodies will be presented, together with preliminary conclusions on the interest of this type of approach.

Department of Immunology, University of Umea. S-90185 Umea, Sweden

Characterization of Con. A-induced suppressive cells, inhibiting TCGFproduction

MARTIN GULLBERG and EVA-LOTTA LARSSON

The cellular and molecular mechanisms regulating TCGF-production, by Con.A-activated spleen cells have been investigated. Pulse experiments demonstrated that TCGF-production in Con.A driven bulk cultures, is arrested after 18-24 h. Neither LAF or macrophages, nor fresh lectin were able to restore the TCGP-production. Cell transfer experiments showed, that 24 h Con. A-activated spleen cells are able to block production of TCGF in fresh culture.~. This

146 14th International Leucocyte Culture Conference, Heidelberg

inhibition was not obtained with soluble factors secreted by the Con.-A-activated cells. The kinetics of appearance of these suppressive cells was found to occur after 18 h, thus induced in parallel to the arrest of TCGF-production in situ. Their induction, but not their effector functions, was radiosensitive. Incubation of 24 h Con.-A-activated cells, in the absence of Con.A, for another 24-72 h, leads to a gradual loss of suppressor activity, that can be recalled by readdition of Con.A, with the same kinetics of appearance as in primary conditions. The Con.A-activated cclls had aftcr a lectin free period of 72 h fully re!>tored thcir capacity to produce TCGF and !>till proliferate in response to Con.A. This together with the finding that no correlation was found between suppressive and cytolytic activity, shows that inhibition of TCGF-production is not due to killing of TCGF producing T cells. Finally, a marked, hut not complete, abrogation of suppressor cell activity was observed in anti-theta and complement treated cultures.

Tel-Aviv University Medical School, Israel, and Stanford University Medical Center, Stanford, CA, USA

The effect of anti-Lyt-2 antibodies on cytotoxic T lymphocyte-target cell binding

NURIT HOLLANDeR, ERIC PILLEMER, and IRVING L WEISSM AN

Anti-Lyt-2 antibodies were shown to block cytotoxic T cells in the absence of complement. Cytolysis of both allogeneic and syngeneic target cells was inhibited at the level of the effector lymphocytes. Proliferation of T cells in mixed lymphocyte cultures and generation of killer cells were also inhibited by anti Lyt-2 antibodies. The antibodies were inhibitory only when present at early stages of the reactions (antibodies had to be added at the onset of the reaction or precoat the responding cells). Concanavalin A-induced proliferation, in contrast to activation by alloantigens, was not inhibited by anti Lyt-2 antibodies. These data suggested that anti Lyt-2 antibodies interfere with initial stages of some antigen specific T cell responses. To analyze the mechanism of inhibition by Lyt-2 antibodies, we studied their effect on binding of cytoroxic T lymphocytes (CTL) to target cells (TC). The recognition stage was followed by: 1. the frequency of CTL-TC conjugates in suspension, 2. the adherence rate of CTL to monolayers of Te. When CTL-TC interactions were performed at 20°C (conditions which enable binding but not lysis of TC), no effect of Lyt-2 antibodies could be demonstrated, although antibodies were bound to CTL at 20°C. However, when CTL-TC interactions were performed at 37°C, Lyt-2 antibodies reduced the rate of CTL-TC binding; the inhibition being at the level of the effector lymphocytes. Under these conditions, antibodies blocked CTL-TC binding both when present in the CTL-TC mixtures, or when preincubated with the effector cells and washed off before incubation of CTL with their targets. Presence of antibodies during the reaction was, however, more efficient in blocking than pretreatment of lymphocytes with antibodies. These observations suggest that Lyt-2 antibodies block a temperature-dependent event involved in CTL-TC interactions and conductive to TC lysis.


lDepartment of Surgery, University of Tiibingen, l Department of Surgery, University of Minnesota, Minneapolis, USA

Effect of specifically sensitized lymphocytes on vascular permeability, regional blood flow and lymphocyte recruitment in sponge matrix allografts

There is evidence that stimulation of specifically sensitized lymphocytes (SSLs) with the specific antigen in vitro results in the production of lymphokines which may influence e.g. vascular permeability (VP), regional blood flow (RBF) and cellular infiltration, when injected in vivo. Whether similar effects can be found in rejecting allografts is still controversial. In addition, it is not clear, whether, similar to macrophages, lymphocytes might be influenced by such factors, too. We studied the effect of SSLs on VP, RBF and lymphocyte recruitment (LR) in a new modification of the sponge matrix allograft model. SSLs were generated in vitro by 5 day culture of BALBlc spleen cells with irradiated (2000 R) allogeneic spleen cells, or in vivo by collecting those cells which had infiltrated a sponge matrix allograft coated with allogeneic cells and implanted into BALBlc mice 14 days earlier. SSLs were injected together with specific or third party allogeneic lymphoblasts into plain sponge matrix grafts implanted s.c. 14 days earlier into BALBl c mice. VP, RBF and LR were studied by i.v. injection of iodine-125-albumine, rubidium-86-chloride and indium-ill labeled lymphocytes, respectively. Compared with the third party graft. VP increased up to 70 % in the specific graft after injection of in vitro generated SSLs and specific allogeneic lymphoblasts. This effect of SSLs wa.~ shown to be immunologically specific. The increase in VP was closely related to the number of SSLs within the graft. RBf also rose significantly in the specific graft, but only by 25 %. No correlation, however, could be found between changes in VP and RBF. Time course studies showed that maximal increase of RBF was 4 hours earlier than that of VP, .mggesting that different factors might be involved. LR to the specific graft was increased as much as 100 %. Thereby the number of SSLs as well as of allogeneic cells within the graft was crucial. SSLs generated in vivo seem to produce similar eHects as those generated in vitro. Killing allogeneic cells before injection into the graft abrogated the capacity of SSLs to increase VP and LR. An immunologically specific interaction between SSLs and cells bearing the sensitizing alloantigen initiates an increase in VP, RBF and LR in the specific allograft. These changes are elosely related to the number of SSLs within the graft. SSLs require living allogeneic cells in order to become active. The strong stimulation of LR to the specific graft might be an important lymphokine mediated amplification mechanism of allograft rejection in vivo.

Central Lab. of the Netherlands Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunology of the University of Amsterdam, The Netherlands

Three functionally distinct subsets of Ty lymphocytes in patients with chronic lymphoproliferative disease

c. J. M. MEUEF, H. J. v. D. REYDEN, H.]. v. D. GRIENO, R.]. M. TJ::N BERGE, A. BOM-V.

NOORLOOS, W. P. ZEl j LEMAKER, A. ASTALDI, M. B. v. 'T VEER, A. E. C. v. O. BORNE

This report shows that four patients with an excess of T lymphocytes bearing Fe receptors for IgG (Ty cells) in blood and bone marrow represent three distinct syndromes, identified by

148 14th Incernational lcucocyte Culture Conference, Heidelberg

clinical presentation and functional as well as cell surface characteristics of the Ty cells. T he three types of Ty ceUs are referred to as Ty l , Ty2 and Ty3. Two paticnts had Ty1 disease, characterized by granulocytopenia a nd recurrent infections of skin and oropharynx. Thc Ty cells from the blood of these patients strongly lysed IgG-sensitized targct cells (K cc ll activity) but did not lyse K562 cells (NK cell activity) and did nOt suppress pokeweed mitogen (PWM) driven Ig synthesis by B lymphocytes (before or after incubation with immune complexes). These Ty cells bound monoclonal antibody against pe riphcral T cells (OKT3) and against T cells mediating cytotoxic andl or suppressor funct ions (OKT8). T hey were OKT4, OKT6 and OKMI negative. The patient w ith Ty2 disease (whose cells wcre kind ly SCnt us by Dr. D. CATOVSKY) presented with recurrent infections of the respiratOry tract, hypogammaglobulinemia and splenomegaly. Those Ty ce lls strongly suppressed PWM driven Ig synthesis but lacked K and NK activity. Their surface phenotype was the same as that of Ty l cells. The patient with Ty3 disease suffered from general malaise. His Ty lymphocytes had strong NK activity and K cell activity bu t failed to suppress Ig production. These cells expressed thc OKTll marker but none of the other OKT markers. In addition they expressed the OKMI marker also found on monocytes. In conclusion the characteristics of the three types of Ty lymphocytes can be listed as fo llows: 1yl : K+, NK- , Suppr- , E+, Fcy+, OKT3+4-6 - S+ 11 ~, OKMI - , OKI-1- . Ty2, r. NK ,Suppr +, E+, fey+. OKT3 ' 4 6- 8+11 +, OKMI - , OKI-l - . Ty3: K I. NK I, Suppr - , E+, Fcy+, OKT3 r6 - S- 11 +, OKMI +, OKI-l +.

It seems likely that the three Ty subsets originated from subsets of Ty cells present in healthy individuals.

Ludwig Institute of Cancer Research, Lausanne Branch , Epalinges, Switzerland, and National rnstitute of Health, Bethesda, M.D., USA

Phenotypic analysis of human cytolytic T l ymphocytes generated in mixed lymphocyte culture

A. M ORETTA, M. C. M INGAR I, A. s. PAUCI, L. M O RETTA , and ].-C. CEROTIINI

Mixed lymphocyte culture (MLC) activated human T lymphocytes express surface markers such as Fc receptors for JgG (FcyR) (1), la-like molecules (2) and 4F2 antigen (3, 4). Al10activated T cells fractionated according to the presence of these markers were analyzed for their cytotoxic capacities. Cell fractionation was performed by rosetting techn iques using ox red blood ceUs (ORB C) coated with anti- la or 4F2 monoclonal antibodies or sensitized with rabbit anti-ORBC IgG. T he various cell fractions were analyzed fo r their specific cytotoxicity against PHA activated target cells bearing the stimulating aUoamigen(s). Fractionation of MLC cells according to su rface FcyR showed that eTL activity was restricted to the FcyR - fraction. Cell separation on the basis of expression of Ia antigens showed no differences in cytolytic activity between positive and negative cells. Additional studies showed that cytOlytic activity was restricted to the subset expressing the 4F2 antigen. 4F2+ cells include up to 30 % FC'{R + cells, whereas FcyR - cells include up to 50 % of 4F2- cells. It can be shown that by us ing fractionation procedures based on the combined use of FcyR and 4F2, CTL activity can be further restricted to the Fcy- , 4 P2+ cell subset. When the same cell populations were analyzed for antibody dependent cellular cytotoxicity (ADCC) aga inst L 1210 mouse cells coated with rabbit antibodies and fo r spontaneous cytotoxicity (NK activity) against K562 human target cells, the cytolytic activity was equally distributed in the la+ and Ia- fractions. However, whereas ADCC was restricted to the FcyR + cell population, NK activity was equally present in the FqR + and FcR - fractions.

1. M O RETTA et al. 1981. Scand. J. Immuno!. (in press). 2. WINCHESTER and K UNHL. 1980. Ad". Immuno!. 2S: 22. 3. ETSF.NBARTH Ct al. 1980. J. Immunol. 124 : 1237. 4. M ORETfA et al. 1981. J. Exp. Med. (in press).


Department of Immunobiology, Wallenberglaboratory, Karolinska Institute, Stockholm, and Department of Cell Research, University of Uppsala, Wallenberglaborawry, Uppsala, Sweden

HLA-DR antigens induce proliferation and cytotoxicity of T cells against haptenated (TNP and FlTC)-self structures

RONALD PALAC10S, GORAN MOLLER, LENA ClAESSON, and PER A. PETERSON

Using anti-HLA-DR antibodies that react against individual chains or the complex of both chains of DR antigens, we have studied the role of DR antigens in activation of T cells and induction of cytotoxic T cells in TNP and FlTC-Iabelled cell cultures, Addition of antisera directed against the heavy, the light or reactive against the complex of both chains at initiation of the cultures strongly inhibited the proliferative response, However, addition of the same types of anti-DR sera 72 h or later after initiation of TNP and FITC-labelled self cell cultures no longer inhibited the proliferative response. T cells from cultures treated with the anti-DR sera were unresponsive to Tnterleukin-2 (11-2). Nonetheless, the anti-DR sera did not inhibit proliferation of T cells that had already acquired sensitivity to IL-2, The anti-DR antibodies abrogated the production of IL-2 in both TNP and FITC-conjugated cell cultures.

Treatment of TNP and FITe-labelled cell cultures with the four different types of anti-DR sera significantly inhibited the induction of cytotoxic T cells. However, addition of the antiDR antibodies at the effector phase of cytotoxicity assays did not inhibit the cytotoxic activity. Killer cells from cultures treated with the anti-DR sera were unresponsive to IL-2 and addition of IL-2 to these cultures only partially restablished cytotoxic activity. In contrast, addition of IL-2 to cultures performed in the absence of the anti-DR antibodies significantly increased the induction of killer cells specific for hapten-conjugated self structures.

Since continuous proliferation of T cells requires of the presence of IL-2 and since resting T cells are unresponsive to IL-2 and unable to absorb the growth factor, we concluded the following: 1. Both the heavy and the light chains of DR antigens participate actively in the activation of T cells by rendering resting T cells sensitive to IL-2 and by inducing production of the growth factor in TNP and FITC-Iabelled cell culrures. 2. The heavy and the light chain play an essential role in the induction of cytotoxic T cells specific for hapten-labelled self structures, most likely, by enabling the.~e cells to respond to

IL-2 and by ind ucing the production of the growth factor. 3. Once T cell s have acquired responsiveness to IL-2 and the growth factor has been produced, there is no further requirement for DR antigens, the availability of Interleukin-2 appears to

determine the expansion and level of hapten-primed cytotoxic cells.

Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

Inhibition of specific T cell mediated cytotoxicity by monoclonal antibodies to human T cell antigens

CHRIS D. PlATSOUCAS and ROJlFRT A. GOOD

Human T lymphocyte subpopulations have been recently defined by monoclonal antibodies, recognizing cell surface antigens selectively expressed on functionally distinct T cell subsets. Monoclonal antibodies of the OKT series define two major subpopulatiom of


peripheral blood T lymphocytes. OKT4-posirive cells (T4 +) are approximately 60% of the peripheral blood E-rosette forming cells and contain cells exhibiting helper or inducer activity. In contrast OKT~ and OKTrpositive cells are approximately 30 % of the peripheral blood Erosette forming cells and contain cells with cytotoxic/suppressor functions. Furthermore the OKT} monoclonal antibody recognizes the majority (90% of the E-rosette positive cells. Anti-T), anti-Ts and anti-Ts monoclonal antibodies inhibited , the absence of added complement, the specific T-cell mediated cytotoxicity at the effector phase against allogeneic targets, in a concentration dependent manner. These antibodies also inhibited, in the absence of added complement, the specific cytotoxicity generated after secondary mixed lymphocyte cultures. Anti-T~ and anti-Ia monoclonal antibodies in the absence of added complement, and control ascites fluid, had no effect on the specific cell mediated cytotoxicity against allogeneic PHA blasts. In contrast neither of these monoclonal antibodies inhibited, in the absence of added complement, the non-spccific cyrotoxicity against the K562 targets, generated in mixed lymphocyte culture. In vitro treatment of effector cytotoxic cells with anti-T) or anti-Ts monoclonal antibodies and complement completely eliminated the specific T cell mediated cytoroxicity but had no effect on the non-specific (natural killer-like) cytotoxicity against the K562 targets, suggesting that natural killer-like cytotoxicity is mediated by a population of cells of non-thymic origin. Anti-T4 and complement treatment had no effect on cithcr thc specific or non-specific cytotoxicity. These results demonstrate that anti-T), anti-T5 and antiT8 monoclonal antibodies, inhibit in the absence of added complement, the specific T-cell mediated cyroroxicity, against allogeneic targets. It is possible that the antigens recognized by these antibodies (TslTR and T1) involve directly or indirectly in the antigen receptor structure(s) or are associated with the lytic mechanism of T cell mediated lympholysis.

Supported in part by Grant CH-151 from the American Cancer Society.

Cleveland Clinic Foundation, Department of Immunology, Research Division, Cleveland, Ohio, USA

In vitro grown T cell lymphomas that express cytotoxic alloreactivity

M. R. PROFFITI and C. DELAMOTIE

We have been studying T cell lymphomas, derived in our laboratory, which may be the malignant counterparts of clones of normal cytotoxic T cells. These lymphomas were derived from C3H/He} mice infected as newborns with Moloney murine leukemia virus. The cells of one of these in viero grown lymphomas (C3HMT1893) exhibit rather specific cytotoxic reactivity when assayed on murine fibroblasts. Grown conventionally (i.e., nOt stimulated with exogenous interleukin 2 and not grown or assayed in the presence of ligands), C3HMT1893 cells preferentially kill BALB/c target fibroblasts; they do not kill identically derived BALB/c targets iofected with MuLV-M. Moreover, they do not kill normal murine fibroblasts of several other allotypes, Con-A-stimulated murine lymphoblast~. or xenogenic target cells. Two clones derived from the parent tumor behave similarly. A different T cell lymphoma (C3HMT1820) displays a different pattern of cytotoxicity on the same spectrum of target cells. The virus itself does not seem to be directly responsible for these phenomena since C3HM'f1893 cells produce little detectable infectious ecotropic MuLV, whereas C3HMT1820 cells actively produce the virus.

14th International Leucocyte Culture Conference, H eidelberg 151

Institute for Medical Microbiology, University of Mainz, 0 -6500 Mainz. FRG

H-2 restriction as a consequence 01 intentional priming. III. Mapping 01 the restriction elements 01 allo-MHC restricted sendai virus or TNP hapten specific CTL-P under limiting dilution conditions

H. STOCK INGER, U. MUNZING, K. PFiZENMAIER, M. ROLUNG HOFF, and H . WAGNER

We have previou.dy shown that within spleen cells ' ) or thymocytes~) of normal mice there exist allo-MH C restricted sendai virus or TNP hapten specific cytotOxic T lymphocyte precursors (CTL-P). This analysis could be performed after selective depletion of alloreactive CTL-P on allogeneic lymphocyte monolayers. The obtained cell population was then stimulated towards the relevant sendai virus infected or TNP hapten conjugated allogeneic stimulator cells. A precursor frequency analysis of these allo-MHC restricted antigen-specific cyto[Qxic T lymphocytes (CTL) revealed a ratio of about 1 to 5 in comparison to syngeneically retricted CTL. The data presented now demonstrate that, similar to self-MHC restricted sendai virus or TNP-specific CTL-P, recognition of foreign antigens o n allogeneic cells is H-2 restricted . Within the majority of the haplotype combinations tested, the restricting clements of both self-MHC and allo-M H C restricted CTL-P mapped to H-2K. D ifferent results were obtained in the H _i D mouse. H ere the CTL response tOwards syngeneic sendai virus infected cells is restricted to H _2Kb. In contrast, allo-MHC (H _2d) restricted sendai vi rus specific CTLP recognized sendai virus in the context of both H-2Kd and H -20d. These data suggest that the restriction phenotype of a responding T cell population is selected as a consequence of antigen presentation.

\. STOCKINGER, H ., K. PrIZENMAIER, C. HARDT, H. ROOT, M. ROLLINC HOFf, and H . WAGNER. 1980. Proc. Nat! . Acad. Sci. USA 77, in press.

2. STOCKINGER, H., R. BARTLETI, K. PFIZENMAIF.R, M. ROl.l.INGHOFF, and H. WAGN I::: R. 198 1, submitted for publicat ion.

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 107, Main7 ..

Inst itute fo r Genetics, Un ivers ity of Cologne, Weye-rtal 121,0-5000 Koln 41, FRG

Functional and biochemical studies on somatic H-2Kk variants

H .-W. VOHR. G. KARMA NN, B. H OLTKAMP, j. NEUER6URG, H. PLOEGH, M. CR AMER , and K. RAJEWSKY

We have previous ly descri bed the isolation of a somatic variant ceUl ine carrying modified H -2Kk molecules. Our way of isolating such variants makes use of pairs of monoclonal anti H -2Kk antibodies, e.g., A an d B wh ich inh ibit each other in binding to H _2Kk and thus react with neighbouring determinants. Cells of a T-lymphomn line expr~ssing H -2 haplotypcs k and d are incubated with antibodies A a nd B, where A is present in a large excess ove r B and B is fluorescein- labelled. Somatic variants which have lost the determinant reactive with antibody A will stain with the labelled antibody B. Such variants are isolated in the Fluorescence Activated Cell Sorter (F ACS). Our method thus permits to select positively for loss variants of - in th is case - H-2Kk surface molecules. At present such sdections of variants were successfully carried OUt for twO antibody pairs yielding cell lines HK13.S1 and HK22.S1.L1.


Biochemical analysis of surface iodinated or biosynthetically labelled variant H -2Kk molecules suggests that the loss of the determinant in alllikelyhood does reflect a structural change in the protein and is not due to a different orientation of the molecule in the cell membrane or to drastically different glycosylation as compared to the parent H-2 molecule . In studies with cytotoxic T-Iymphocytes (CTL) we found that TNP-specific H_2Kk restricted CTLs can discriminate one variant (HK13.S1) but not the other one (HK22.Sl.Lt ) from wild type. This can be shown in direct cytolysis and in cold target inhibition experiments. Allon:active H-2Kk specific eTLs stimulated in bulk cultures are unable to discriminate variant from wild type cells. Further studies are under way to analyse the variants in additional H-2Kk restricted systems.

Department of Surgery and VA Hospital, University of Alabama in Birmingham, Birmingham, AL 35294, USA

Evaluation of complement receptor bearing cortisone resistant thymocytes for IgG or IgM Fc binding

A. s. W AUA and E. W. LAMON

Mice wert: injected with cortisone acetate following which their thymocytes were tested for receptors for the third component of the complement (C3). An erythrocyte-antibodycomplement (EAC) rosette assay was used to detect C3 receptors. Our previous studies have shown that cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18 % at day 7. In our present studies, complement receptor bearing cortisone resistant thymocytes were evaluated using a mixed rosette experiment with double and triple marker studies. Chicken red blood cells (CRBC) were coated with mouse anti CREC IgG. Ox red blood cells (ORBC) were first treated with fluorescein diacctatc and then coated with mouse anti-ox IgM. Sheep red blood cells (SRBC) were first coated with rabbit anti sheep IgM and then with fresh A/J serum (CS deficient). All these indicator cells were then mixed together with thymocytes obtained from normal and cortisone injected animals. IgG Fc receptor bearing cells were differentiated from JgM Fc and C3 receptor bearing cells by the size and shape of CRBC (nucleated, football shaped and much larger than ORBC or SRBC). IgM Fc receptor bearing cells were examined under the fluorescence microscope and thus the C3 receptor bearing cells were counted as such. The mean of four experiments showed that 5 % of C3 receptor positive cortisone resistant thymocytes were positive for IgM Fe binding whereas 3 % of C3 receptor positive cells were positive for IgG Fe binding also. 8 % of cortisone resistant thymocytes were positive for C3 receptors alone.

Supported by VA Hospital project # 5132-03 and grant II I-F-32-CA0629S-03, CA!3148, CA I 7273-05 and CA26257-0t from the National Institute of Health, USA.

Central Lab. of the Netherlands Red Cross Blood Transfusion Service and the Lab. of Exp. and Clin. Immunology of the University of Amsterdam, The Netherlands

Two types of specific suppressor cells in hum.n mixed lymphocyte reactions

w. P. ZEljLEMAKER, B. HUIS, H. C. ROMKE, and P. P. ROBERTSON

Lymphocytes from 4 individuals possessed specific suppressor T cell activity, inhibiting the response in MLR of only certain allogeneic cells. Two types of suppression were observed:


1. In 3 individuals from 2 families, the suppressor cells showed specificity for only certain allogeneic responder cells. The susceptibi lity to suppression segregated independently of HLA. Similarly, the occurrence of the suppressor cell activity segregated in families independently of HLA. Finally, the lymphocytes from the .. suppressor~ donors showed a strong response when stimulated by susceptible cells which they suppressed in the reverse situation. 2. A second type of suppressor cell strongly inhibited the MLR response of lymphocytes from all HLA-Al or A!~-positive individuals, but not of those from any other individual.ln further contrast to the first type, these suppressor lymphocytes did not respond in MLR when stimulated with «susceptible .. cells, i.e. those expressing HLA-A2 or An. Again the occurrence of suppressive activity segregated independently of HLA. In spite of the differences in specificity, these two types of suppressor cells had several properties in common; a) they occurred spontaneously in healthy, non-transfused males. Suppression was consis

tendy demonstrated for at least 3 years; b) the suppressor activity was associaled with T cells expressing a receptor for IgG (i.e. 1'y

cells); c) whereas mitomycin-treated lymphocytes suppressed, y- irradiation abolished suppressive

activity in a dose-dependent way; d) the suppressor cells inhibited the response of «susceptible ., lymphocytes to any allogeneic

stimulator cell, as well as the gene ration of cytotoxic T l ymphocytes, bur not the response to T cell mitogens such as PH A or ConA;

c) The !\uppressor cells strongly inhihited the production of IgM and IgG by «susceptible» cells when cultured in the presence of pokeweed miragen (PWM).

The suppressive activity was not cau.~ed by direct cytotoxicity. Prostaglandins appeared to be not involved. Soluble suppressor factors could not be demonstrated, although inhibition of protein synthesis tended to decrease suppres.~or activity. These data indicate rh at Ty cells can playa regulatory role in Ml.R, and can in this respect show specificity at the level of the responder cell.

Partly supported by grant CS5 of the Dutch Kidney Foundation.

t lymphocytes: effector functions

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