Workshop H T Cell Receptors, T Cell Markers

WorkshopH T Cell Receptors, T Cell Markers

Institut für Immunologie der Joh. Gutenberg Universität, D-6500 Mainz, FRG

H.l Antigen-presenting bone marrow macrophages interact selectively with distinct T cell dones

H. G. FISCHER and A. B. RESKE-KuNZ

Bone marrow-derived macrophages (BMM<I», differentiated in vitra in the presence of L-cell CSF, are known to exert antigen-presentation function following activation by interferon-y. When the responsiveness of various T ceillines to antigen presented on Iymphokinetreated BMM or spleen cells was compared, BMM were not capable of inducing antigenspecific proliferation of all T ceillines, suggesting distinct activation requirements of different T cells (1). Several T cell clones were not stimulated by BMM within the first 14 days of subculture in IL 2-containing medium after antigenic challenge on spleen accessory cells, but developed increasing reactivity towards BMM as antigen-presenting cells upon prolonged culture in IL 2. During this culture period, these T cells developed increasing sensitivity towards antigen. Other T cell clones were not activated by BMM even after protracted culture in IL 2. A proliferative response of these non-reactive T cell clones could not be achieved by the addition of cytokines such as IL 1, nor by supplying aHogeneic spleen ceHs or irradiated BMM-reactive T ceHs. When presented on splenic accessory ceHs, these T ceHs required relatively high doses of antigen. These data suggest that efficient T cell activation depends on high avidity T cell-BMM-interaction. On the other hand, the stimulatory activity of antigen-presenting BMM was accompanied by an inhibitory effect on the proliferation of some T cell clones. This inhibition depended on the presence of the specific antigen.

1. FISCHER, H. G., A. B. RESKE-KuNZ, E. SPÄTH, and E. RÜDE. 1985. Eur. J. Immunol. 15: 957.

Supported by the Deutsche Forschungsgemeinschaft, SFB 311.

Department of Medical Microbiology and Immunology, University of Ulm, FRG

H.2 Staphylococcal enterotoxins: MHC dass II-dependent probes for the T cell receptor for antigen

B. FLEISCHER and H. SCHREZENMEIER

Staphylococcal enterotoxins (SE) are the most potent mitogens for T Iymphocytes that are known. Concentrations of less than 1O-9 M are sufficient for T ceH activation. We have used

XIXth Meeting of the Society of Immunology . 329

cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by SE. Although CD4+ and CD8+ T cell as well as CD4-8- NK clones respond with cytotoxicity and/or proliferation to SE, for triggering of all these clones, the presence of MHC class Ir molecules on accessory or target cells is necessary. This requirement for class Ir antigens is reflected by selective binding of SE to class Ir molecules. Furthermore, T cell activation by SE shows clonal heterogeneity due to differential binding of SE to the clonotypically expressed T cell receptor. We conclude that SE are functionally bivalent mitogens binding highly selectively to HLA class Ir molecules and the T cell receptor. Thus, compared to other polyclonal T cell activating agents, activation with SE most closely resembles the physiological way of MHC-restricted antigen recognition by T lymphocytes.

Institut für experimentelle Immunologie, Philipps-Universität, 3550 Marburg, FRG

H.3 Successive changes of the cellular composition in the fetal and neonatal thymus of MRLllpr mice, investigated in cryosections

S. GEHRMANN and K. U. HARTMANN

MRL/MP-Ipr/lpr mice spontaneously develop lymphoproliferation and autoimmunity during the first 8-12 weeks of age; an expansion of an unusual T-Iymphocyte subpopulation (Lyt-l( +), Lyt-2( -), UT4( -), B 220( +)) seems to be an early sign of the beginning disease. These cells develop within the thymus; neonatal thymectomy is known to prevent the onset of lymphoproliferation. - We have stained cryosections of the developing thymus (from day 14 of gestation till after birth) with a variety of monoclonal antibodies directed against surface antigens of thymocytes or stroma cells, isolated after immunization with fetal thymus of nonauto immune mice. We shall report about sequential changes of the thymocytes during normal development and the appearance of the unusual T-Iymphocyte subpopulation in lpr mice.

Department of Medical Microbiology and Immunology, University of Ulm, D-7900 Ulm, FRG

H.4 T cell antigen receptor ß-chain gene re arrangements in nu/nu mice

R. HAARS, 1. MILTNER, J. REIMANN, and H. WAGNER

Rearrangement and expression of the T cell antigen receptor and the y-genes during T cell ontogeny is a regulated process. These gene re arrangements occur primarily or exclusively in the thymus, although some gene re arrangements occur outside the thymus in fetal li ver cells that may be committed T cell progenitors (R. HAARS et al., 1986). This finding led us to investigate the extent of T cell antigen receptor ß-chain gene re arrangements in different lymphoid tissues from athymic nu/nu mice. Several conclusions can be drawn from this work. First, Southern blot analysis clearly demonstrates Dß2 and Jß2 gene re arrangements in the liver, bone marrow and spleen of adult nu/nu mice of different age. This result indicates for the first time that ß-chain gene re arrangements can occur outside the thymus. Second, although Dß2 and Iß2 gene rearrangements are present in the spleen of athymic mice, we do not observe any germline signal using these gene segments or an IgVk gene segment as a probe. Thus we

330 . XIXth Meeting of the Society of Immunology

canclude that Dß2 and Jß2 gene re arrangements in the spleen of nu/nu mice frequently involve de!etions of these gene segments as weil as the rest of chromosome 6. Third, the size of most of these Dß2 and Jß2 gene rearrangements is not consistent with the size expected from DßrJß2 gene re arrangements which leads us to suggest that most of these ß-chain gene rearrangements are aberrant. Fourth, analysis of ß-chain gene rearrangements in lymphoid tissues of individual mice of different age shows no simple carre!ation between the age of the mice and the status of ß-chain gene rearrangements. Together the results indicate that T ce!1 differentiation in nude mice is clearly retarded and not synchronized in individual mice of the same age. The apparent lack of functional T cells in athymic mice might be in part due to defective T ce!1 receptor ß-chain gene rearrangements.

Dept. Immunology, I. Med. Klinik, Univ. Krankenhaus, 2000 Hamburg 20, FRG

H.5 Selective regulation of lymphocyte homing receptors with differing organ specificity upon activation

A. HAMANN, D. JABLONSKI-WESTRICH, R. HARDER, and H.-G. THIELE

Lymphocyte homing into different organ systems of the body is directed by specific homing receptors on Iymphocytes and corresponding recognition sites on high endothe!ial venules (HEV). Whereas virgin Iymphocytes seem to possess multiple binding specificities, for blasts and memory cells very se!ective migration patterns are found. To better understand the mechanisms regulating the homing of such populations, we have analyzed Iymphocytes stimulated by different mitogens for the expression of the putative homing receptor gp 90-Me! 14 and for their ability to bind to high endothelial venules (HEV) in vitra, with the following results: 1) Submitogenic stimuli induce an increase in homing receptor gp 90-Me! 14 expression. This is true for alm ast alllymphocytes in autologous cultures, for the not fully activated fraction of cells in periodate, LPS or Con A -treated cultures and for cultures stimulated with suboptimal doses of Con A. 2) In general, full blast transformation is associated with a decrease or complete loss of gp 90-Me! 14 expression. This applies to all activating systems used, but only for the major part of the Iymphocytes; indeed more than 30 % of fully activated cells may express very high leve!s of the Mel 14 antigen. 3) Con A and periodate stimulation lead to a selective and nearly camplete suppression of the capacity to bind to HEV of Peyer's patches (in the frozen section assay), under canditions, where binding to peripheral nodes is increased. This indicates a differential regulation of the two respective receptors, the mucosa-system specific homing receptor being down-regulated very early in these activation system.

I Max-Planck-Institut für Immunbiologie, Stübeweg 51, and 2 Universitätsklinik, Abt. Rheumatologie, Hugstetter Str. 55, 7800 Freiburg, FRG

H.6 A study on the expression and structure of T cell receptor genes from patients with autoreactive arthritis

A. HINKKANEN1, V. STEIMLE1

, H. H. PETER2, M. SCHLESIER2, and J. T. EpPLEN1

In order to e!ucidate the role of autoreactive T cells (Tcs) in rheumatic diseases we are studying the expression and structure of the Tc receptor (TcR) a and ß genes of a highly


specific, DR-dependent helper Tc clone UA-S2 isolated from the synovial fluid of a patient (UA) with post-rubella-arthritis (1). DNA sequence analysis of the TcR ß gene (lambda phage clone 335) revealed a productive rearrangement (Vß - Dß2.1 - Jß2.3 - Cß2 with several N nucleotides). There was no extensive homology to the Vß element from a c1ass II-autoreactive murine Tc clone (2). In the 5' untranslated region of 335, two conserved tandemly organized 26 basepair repeats were detected. These were highly homologous (85 %) to each other as weil as to a nuclear protein-binding domain of a TcR Vß gene from another human Tc line (3). All three sequences carry the motif AGT GAT at their 5' end. The relevance of these signal sequences for expression is under study.

The TcR a gene of UA-S2 is expressed in fulllength and for its isolation we are currently constructing a c-DNA library. Moreover, using specific oligonucleotide probes we are screening a panel of autoreacvtive Tc clones from rheumatic patients for TcR-a and -ß rearrangements. Preliminary hybridization data indicate that several other autoreactive T cell clones from the patient UA do not use the same TcR ß gene. Hybridization and sequence analysis data will be reported and discussed. 1. SCHLESIER, M., e. RAMB-LINDAUER, A. E. HINKKANEN, J. T. EpPLEN, H. H. PETER. 1986.

Immunobiology 173: 345. 2. HINKKANEN, A., B. KEMPKES, H. STOCKINGER, H. U. WELTZIEN, J. T. EpPLEN. 1987. In: T

Cell Receptor. UCLA Symposia on Mol. and Cellular Biology Vol. 73, J. KappIer and M. Davis (eds) The Alan R. Liss. Inc., New York, NY.

3. ROYER, H. D., E. L. REINHERZ. 1987. Proc Natl Acad Sci 84: 232.

Immunologisches Labor, Medizinische Univ.-Klinik, Abt. II, D-7400 Tübingen, FRG

H.7 Rearrangement and transcription of T ceH receptor genes in functionaHy distinct human T ceH clones

F. KALTHOFF, G. PAWELEC, and P. WERNET

Human helper T cell clones (TCC), derived by limiting dilution from allosensitized Iymphocyte populations, were characterized by their IL 2-driven autocrine proliferative capacity and by supporting B cell differentiation when stimulated with the relevant alloantigens (phase I TCC). After about 35 population doublings (PD), however, greater than 90 % of such helper TCC lost these functions and instead acquired strong antigen non-specific suppressive activity (phase II TCC). In addition, about one third of these phase II TCC displayed NK-like activity. Surface marker phenotypes of phase land II TCC always displayed CD4, CD3 and WT 31 positivity. We wanted to investigate whether such modulation of functions could be accounted for by changes in antigen receptor specificities or altered transcriptional status of T cell receptor (TCR) alpha, beta or gamma chain genes. Southern blotting analyses of TCR alpha, beta and gamma chain genes c1early confirmed monoclonality for all helper TCC and demonstrated the same re arrangements for phase II as compared to phase I TCe. Northern blotting showed expression of TCR alpha and beta but not gamma chain genes for all helper TCC regardless of their functional status. However, in certain cases heterogeneity was observed with respect to the relative proportion of full-Iength, complete or shorter-sized, incomplete messages for TCR beta chain genes. In contrast, one CD4 j , CD3 +

but WT 31- TCC with constitutive NK-like function expressed a full size 1.7 kb message for the TCR gamma chain but no alpha chain mRNA and only a shorter-sized 1.0 kb message for the TCR beta chain. Data thus far indicate that 1) there are no obvious differences between phase I and phase II TCC in the arrangement and transcription of TCR alpha and beta messages and 2) NK-like activity associated with either phase II TCC or WT 31- TCC is not correlated with the expression of a particular pattern alpha, beta or gamma TCR messages.


Max-Planck-Institut für Immunbiologie, Stübeweg 51, and ". Junior Research Unit, MaxPlanck-Institut für Immunbiologie, 7800 Freiburg, FRG

H.8 Receptor repertoire studies in TNP-specific T cell responses in C57Bl/6 mice

B. KEMPKES, K. EICHMANN, J. T. EpPLEN"·, and H. U. WELTZIEN

Recently, limited heterogeneity of expressed T cell receptor (TCR) a- and ß-chain genes in TNP-specific cytotoxic T cells (CTL) was reported (1, 2). Sixteen out of 42 CTL clones expressed an identical a/ß-TCR combination as determined by oligonucleotide hybridization in RNA- and DNA-gels and blots. It was not clear, however, whether this major CTL clonotype was due to selection in vitro or in vivo, since all of these clones had been obtained from CTL lines after 6-10 weeks of culture in vitro. Therefore, clones were established after only two bulk-stimulations of spleen cells in vitro from TNP immunized mice (one immunization). The same protocol was used to obtain CTL-clones from repeatedly immunized mice (five immunizations). Only those clones were investigated on the molecular level that originated from different mice, i.e. that were independent. Preliminary results from RNA and DNA analyses indicate, that both classes of clones appear less homogenous than the published collection (1, 2). Nevertheless, two out of eleven clones appear to have identical TCR ß-gene rearrangements: both clones use the Jß2-6 and Cß2 segments in the fulllength mRNA and show identical patterns of rearrangements in Southern blot hybridizations with different restriction enzymes. These data suggest that the predominant clonotype observed earlier is probably due to selection in vitro and not due to over representation of the specific TCR in vivo.

1. HOCHGESCHWENDER et al. 1986. Nature 322: 376-378. 2. HOCHGESCHWENDER et al. 1987. Nature 326: 307-309.

Supported by DFG grant We 379/6-1.

1 Memorial Sloan Kettering Cancer Center, N ew Y ork, and 2 Hunter College of the City University of New York, U.S.A.

H.9 A transformation-associated 130 kD cell surface glycoprotein is growth controlled in normal human cells

C. E. KLEIN2, H. L. OZER2

, F. TRAGANOSl, J. ATZPODIEN1, and L. J. OLD1

Growth-controlled expression in normal cells and transformation induced over expression are two characteristics reported for molecules that appear to be critically involved in the promotion of cell proliferation. In order to identify new gene products that might play an important role for growth control in the normal and transformed state, we have utilized these criteria to test more than 100 monoclonal antibodies originally developed in our laboratory against cell surface antigens of human tumor cells. Synthesis and cell surface expression were studied in radioimmunoprecipitations and in immunofluorescence tests using a flow cytometer, respectively. By this approach, we have identified a 130 kD cell surface glycoprotein (gp 130) fitting the model of a molecule relevant to cell proliferation i~ diverse cell systems. Synthesis and cell surface expression of gp 130 was highly increased in viral (SV 40) and chemical (4-Nitroquinolineoxide) transformed human fibroblasts, when compared to the normal parent cells. Gp 130 was also highly expressed in 3/3 fibrosarcoma cell lines, skin


derived squamous cell carcinoma- and T celileukemia ceillines. In contrast, gp 130 was not detectable or in low levels on normal adult fibroblasts, keratinocytes and resting T cells. Furthermore, gp 130 was induced upon T cell activation and serum stimulation of fetal fibroblasts. Bivariate analysis of DNA- and cell surface immunofluorescence suggested that the increase of gp 130 expression after serum stimulation is associated with the transition of cells from Go to GI'

Westfälische Wilhelms-Universität, Institut f. Immunologie, Münster, FRG

H.lO Expression of Mls determinants on aT cell clone: characterization of the cellular immune response to this clone

S. KONIGORSKI and R. DI PAUL!

An established murine T cell clone could in du ce a primary proliferative response in vitro by spleen cells from different mouse strains. The T cell clone (CBA-4) of CBA/J(H-2k,Mls') origin, induced a response in spleen cells syngeneic at the H-2 and differing from CBA-4 at the Mls. The clone could also induce a response in H-2 allogeneic spleens. From the primary anti CBA-4 cultures, we could establish ceillines. Some of the lines were specific for Mls' or Mlsd

in the context of H-2k and H-2d but not of H-2Q• The ceillines were also stimulated by F1 cells

between H-2d/Mlsb and H-2Q/Mls'. Celilines originating from H-2 allogeneic spleens were specific for cells expressing H-2K,Dk

. The primary response to CBA-4 was dependent on Ia+ splenic adherent cells and could be blocked by monoclonal antibodies to I-A, I-E and to H-2K determinants. The ceillines, specific for Mls"d and H-2k, were blocked only marginally by anti I-A, I-E, H-2K specific for the H_2k haplotype. In contrast, nearly complete inhibition was obtained with monoclonal antibodies to I-A and I-E on H-2d/Mls' (DBA/2) cells. We would like to suggest that the Mls molecule is closely linked on the membrane to MHC molecules, and that it plays an accessory role in the activation of T cells.

Forschungsinstitut BorsteI, D-2061 BorsteI, FRG

H.tt Expression of dipeptidyl peptidase IV (DPPIV) on human T lymphocytes after mitogenic and antigenic stimulation

T, MAITERN, W. SCHOLZ, H.-D, FLAD, and A, J. ULMER

Dipeptidyl peptidase IY (DPPIY) is an ectoenzyme found in the plasma membrane of Tlymphocytes, In former experiments, we have found that the expression of DPPIY on T cells is associated with the capacity to produce IL 2 and to proliferate after stimulation, Furthermore, we have shown that mitogenic stimulation of human MNC with PHA, Con A or PWM resulted in an enhancement of the number of DPPIY+ cells from 40 % to over 80 %. The aim of this study was to investigate wh ether this increase is due to a de novo expression of the enzyme, DPPIY+ and DPPIY- T cells were isolated by cell sorting and stimulated with PHA or tetanus toxoid and cultured under limiting dilution conditions in the presence of irradiated feeder cells (MNC). We found that after mitogenic stimulation the frequency of proliferating cells is nearly 1/1 seeded DPPIY+ as weil as seeded DPPIY- ceiL After antigenic stimulation,

334 . XlXth Meeting of the Society of lmmunology

we found a frequency of about 1/2400 seeded DPPlV+ cells and about 1/6700 seeded DPPlVcells. All tested clones were found to contain 40 %-95 % DPPlV+ cells. The proliferation of DPPlV- T cells and the expression of DPPlV by these cells may depend on DPPIV+ T ceHs, since DPPlV- T cells proliferate in the presence of DPPlV+ feeder cells. We have also investigated whether cytokines such as lL 1, lL 2, lFNy and TNFa are involved in the de novo expression of DPPlV. Unsorted T cells, DPPlV+ and DPPlV- sorted T cells were activated with PHA and cultured in the presence or absence of the various cytokines. The results show that these cytokines did not alter the mitogen stimulated expression of DPPlV. We conclude that 1) DPPlV is expressed de novo after mitogenic or antigenic stimulation. 2) All tested cytokines have no influence in the mitogenic stimulation of expression of DPPlV.

Dept. of Med. Microbiology and lmmunology, University of Ulm, D-7900 Ulm, FRG

H.12 T cell receptor ß-gene rearrangements in fetalliver cell populations of C57Bl/6 mice

I. MILTNER, H. WAGNER, and R. HAARS

T cell precursors arise from hematopoietic stern cells which reside in the fetal li ver or in the adult bone marrow. These precursors leave the fetal liver at about day 11 of gestation and mi grate to the thymus where they proliferate and differentiate. The rearrangement and expression of T cell receptor genes are clearly an important step in T cell differentiation. Analysis of T cell receptor gene segment assembly during T cell ontogeny has revealed that this process is primarily restricted to the thymus. Although some y-gene rearrangements have been demonstrated to occur outside the thymus in fetal liver cells (R. HAARS et al., 1986), the commitment of T cell precursors in the fetal liver in terms of ß-gene rearrangements has, however, not been established. Therefore, we separated fetalliver cell suspensions of C57Bl!6 mice of different age on discontinuous BSA gradients. So far, Southern blot analysis of the DNA obtained from the four fractions A, B, C and D revealed ß-chain gene re arrangements in day 12 fetalliver. These ß-chain gene re arrangements do not involve the first cluster of the ßchain locus, namely DßI> Jßt or Cßt but appear to be restricted to Dß2 and Jß2 depending on the restriction enzyme digest. The size of these Dß2 and Jß2 gene segment re arrangements is however not consistent with the size of known Dß,-Jßz gene rearrangements. Therefore, we have cloned some of these rearrangements and are in the process of sequencing. Together, these results clearly demonstrate for the first time that ß-gene rearrangements can occur outside the thymus in fetalliver cells.

lnst. of lmmunology, Univ. of Heidelberg, D-6900 Heidelberg, FRG

H.13 Characterization of guinea pig T cell receptor cDNA clones using an expression library

J. SCHENKEL, H. SCHÄFER, R. SCHÄFER, U. BARON, and R. BURGER

The u- and ß-subunits of the T cell receptor for antigen contain constant regions which are encoded by a single (a) or two closely related genes (ß). Antibodies to constant region


determinants would provide a valuable tool for analysis of T cell function and differentiation both at the level of T cell populations and at the clonallevel. We used the cDNA probes TT11 and 86T5 encoding segments of the murine T cell receptor u- and ß-chain for analysis of the guinea pig T cell receptor. The murine cDNA probe crosshybridized in Southern blots with genomic guinea pig DNA. They were used for the screening of a guinea pig /-gdl cDNA library. The library was prepared from the poly(A) containing RNA of guinea pig Con Aactivated T cell blasts. Several cDNA clones were isolated. These dones contain inserts of about 1 kb. They hybridize in Southern blots with the corresponding mouse cD NA probes, suggesting that the analogous guinea pig genes are present. Clone 170 representing presumably the guinea pig ß-chain equivalent was further characterized. The restriction sites show a high similarity to the murine 86T5 ß-chain probe. The corresponding guinea pig gene showed rearrangement in the course of T cell differentiation. This was shown by differences in the restriction fragments of DNA from a guinea pig T cell clone compared to genomic DNA isolated from macrophages or liver cells. In contrast, DNA from a population of peripheral T cells did not show this discrete restriction pattern as one would expect because of their polyclonal nature. In Northern blot analysis hybridization of T cell RNA with clone 170 occurred in the range of about 1.5 kb. The hybrid pro teins encoded by the murine and guinea pig T cell receptor were expressed in E. coli. The hybrid proteins contain ß-galactosidase or MSII polymerase as carrier moiety. Rabbits and rats were immunized with the hybrid pro teins purified by preparative SDS-PAGE. Antisera and a monodonal antibody were obtained which react with the hybrid protein in the bacterial Iysate. The antibodies are used for serological assays with guinea pig T cells and immunoprecipitation.

Supported by DFG Bu 400/2.

Department of Hematology, Oncology, and Immunology, Robert-Bosch-Krankenhaus, Auerbachstr. 110, D-7000 Stuttgart 50, FRG

H.14 Loss of cryptic Thomsen-Friedenreich antigens du ring mitogenic activation of human T and B lymphocytes

H. R. SCHMITT, M. F. WOLF, and K. SCHUMACHER

Thomsen-Friedenreich (TF) antigens are cryptic carbohydrate determinants on erythrocytes and Iymphocytes that can be exposed on tumor cells without terminal sialic acid. Recently, their role in organotropic metastasis was demonstrated. Furthermore, exposure of TF antigens on Iymphocytes by neuraminidase treatment results in an accumulation of Iymphocytes in the liver.

A monoclonal antibody (49H8) was used to investigate TF antigen expression during Iymphocyte activation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM). The monodonal antibody is highly specific for phenyl-ß-galactoside. Eighty % of purified T cells from peripheral blood and 93 % of purified B cells were found to carry cryptic TF antigens. During 5 days of stimulation with PHA, the number of blast cells (8 !Am) in the cultures increased from 1 % to 85 %. At the same time, TF dererminants carrying blast cells were reduced to 13 % of the cells at day 5 of culture. PWM stimulation resulted in 48 % of blast cells after 5 days, with none of the blast cells being positive for TF antigens.

With regard to the accumulation of TF expressing Iymphocytes in the liver, these data suggest, that the loss of TF antigens on activated Iymphocytes may keep these cells in the circulation, while the presence of cryptic TF antigens on resting Iymphocytes may result in homing or degradation of cells.

Supported by the Robert-Bosch-Stiftung.


Department of Medical Microbiology and Immunology, University of Ulm, D-7900 Ulm, FRG

H.tS TNP-specific, H-2 restricted Lyt2+ lL 2 producing T lymphocytes clonally segregate from L yt2+ cytotoxic T lymphocytes

J. SCHMITT, H. WAGNER, and K. HEEG

We recently reported that cell sorter purified Lyt2+ T cells can be induced upon stimulation with TNP-modified syngeneic stimulator cells to proliferation, IL 2 secretion and cytotoxicity independent of L3T4+ T cells. We determined the frequencies of TNP-specific Lyt2+ IL2-producing T cells to be in the range of 1/600 and demonstrated a specificity and restriction phenotype as exquisite as that of Lyt2+ TNP-specific CTL. We have here analyzed whether the two functions (IL 2 secretion and cytotoxicity) are mediated by bifunctional Lyt2+ T cells or by distinct functional sub sets within Lyt2+ T Iymphocytes. To this, we have set up limiting dilution cultures with a high (> 95 %) probability for clonality and tested the TNP induced cultures in a split culture analysis for IL 2 secretion and cytotoxicity. The analyses revealed that the majority of growing colonies displayed either an IL 2 producing phenotype without cytotoxic activity or a cytotoxic phenotype without IL 2 secretion. Only a few colonies « 5 %) seemed to be bifunctional. Thus, these data parallel similar results in allo Class I MHC responses of Lyt2+ T cells and provide evidence for T-T cell interactions within the Lyt2+ T cell subset in the generation of hapten specific H-22-restricted cytotoxic effector cells.

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Workshop H T Cell Receptors, T Cell Markers