- Email: [email protected]
E-2: Receptors Involved in T Cell Activation
Workshop E-2 Receptors Involved in T Cell Activation
I.C.R.F. Human Tumour Immunology Group and Department of Clinical Haematology, School of Medicine, University College London, London WC1, U.K.
366. Human T lymphocyte activation investigated using mitogenic monoclonal antibodies P. C. L. BEVERLEY, D. WALLACE, E. D. ZANDERS, K. O'FLYNN, and D. LINCH
Antibodies to the T3-receptor complex of human T lymphocytes may be mitogenic. While the IgG2a anti-T3 antibody OKT3 stimulates peripheral blood mononuclear cells (PBM) of all normal donors, the IgG1 antibodies UCHTl and Leu 4 stimulate PBM from only 70 % of individuals. The response of non-responders can be reconstituted with accessory cells from responders (1). This system provides a model for investigation of accessory cell-T cell interaction. We have used the calcium sensitive dye Quin-2 to investigate the effects of antibodies to the T3-receptor complex and have shown that UCHTl can trigger a calcium flux in both responder and non-responder lymphocytes confirming both that non-responder T cells react «normally» to ligand binding to T3 and that a second signal provided by accessory cells is required for initiation of DNA synthesis in T cells. In further experiments we have attempted to define what is provided by the accessory cells using agents which crosslink the anti-T3 antibody or stimulate or replace the accessory cells. In addition we have attempted to define to what extent the method of accessory cell activation regulates the subset of T cells activated or the pathway of differentiation of the activated cells (2). 1. TAX, W. J. M., WILLIAMS, H. W., REEKERS, P. P. M., CAPEL, P. J. A., and KOENE, R. A. P. Polymorphism in mitogenic effect of IgG1 monoclonal antibodies against T3 antigen on human T cells. Nature 304: 445--447 (1983). 2. VAN WAUWE, J. P., GOOSSENS, J. G., and BEVERLEY, P. C. L. Human T lymphocyte activation by monoclonal antibodies: OKT3, but not UCHTl, triggers mitogenesis via an interleukin 2-dependent mechanism. J. Immunol. in press (1984).
Department of Experimental Pathology, St. Mary's Hospital Medical School, London, U.K.
367. Demonstration of a third component of the human T cell antigen complex A. W. BOYLSTON, R. D. GOLDIN, and C. S. MOORE HPB-All is a human T cell leukaemia line which expresses a clonotypic molecule with all the properties of the putative T cell antigen receptor, i.e., comodulation with the T3 antigen, heterodimeric structure with. 50,000 and 40,000 molecular weight subunits; and strict
242 . 16th International Leucocyte Culture Conference, Cambridge clonotypy. Whilst investigating other McAbs raised against HPB-All a McAb (A5) with the ability to comodulate the TCLIi antigen of HPB was discovered which has a structure different from TCLIi and T3 but which has properties suggesting that it is also part of the antigen receptor complex. These are: 1) A5 comodulates T3 and TCLIi on HPB-All, 2) A5 comodulates T3 on normal T cells, 3) A5 is mitogenic for normal T lymphocytes, 4) A5 has the same cell type and cell line distribution as T3, 5) A5 recognizes a molecule different from T3 or TCLIi because it precipitates a membrane component with a MW of 64,000 in SDS-PAGE under both reducing and non-reducing conditions.
Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London, WC ItE, 6BT, U.K.
368. Studies on the human T cell receptor for antigen M. K. COLLINS and M. J. OWEN Recently a mouse eDNA clone, 86Tt, which codes for a T-lymphocyte specific molecule with an immunoglobulin like structure was isolated. We have used this clone as a probe to investigate the structure of homologous molecules in human T-lymphocytes. In Northern blot analysis 86Tl detected the presence of an analogous mRNA in various human T cell lines and in functional T helper cell clones, but not in any other cell type. 86Tt was then used to isolate a eDNA clone from a library constructed in )"GTto from the T4 positive human T cell line J6. The structure of this clone will be discussed.
Department of Immunology, University of Ulm, FRG
369. Involvement of the T3 antigen in polyclonal T cell activation by mitogenic lectins and oxidation B. FLEISCHER Accessory cell (AC)-dependent mitogen-induced polyclonal T cell activation has often been used as a model for antigen-specific triggering, the precise mechanisms, however, by which T cells are triggered by mitogens, are still a matter of debate. We have recently shown that mitogenic lectins or the introduction of aldehydes into cell surface glycoproteins by oxidation activate T cells by crosslinking T cell membrane proteins with the AC surface (1). In the present study, the effect of monoclonal antibodies against the T cell surface antigens T3 and T4 on AC-dependent mitogen-induced proliferation of human 14+ T cell clones and purified peripheral blood T cells was studied. To avoid the Fe-receptor dependent mitogenic effect of the OKT3 antibodies, monocytes were replaced by Fe-receptor negative tumor cells as AC. Monoclonal antibody OKT3 but not OKT4 inhibited the response of both T lymphocyte clones and purified T cells to mitogenic lectins and oxidation. The inhibition was not due to nonspecific effects of the monoclonal antibody since it affected only the initial triggering but not the proliferation of activated T cells and it could be overcome by higher concentrations of lectin. Furthermore, OKT3 antibody at the same concentration that was inhibitory in this system was mitogenic in the presence of Fe-receptor bearing monocytes. Surface modulation of T3 but not T4 antigen led to unresponsiveness to a mitogen pulse given directly after modulation. These findings suggest that antigen-specific and mitogen-induced T cell triggering are due to the interaction with the same receptors of the T lymphocyte. 1. FLEISCHER, B. 1983. Eur. J. Immunol. 13: 970.
16th International Leucocyte Culture Conference, Cambridge· 243 Stanford University School of Medicine, Stanford, CA 94305, U.S.A.
370. Sequence Organisation of a T cell receptor gene N. R. J. GASCOIGNE, Y.-S. CHIEN, D. M. BECKER, NADINE E. LEE, J. KAVALAR, and M. M. DAVIS
We have recently described the isolation and characterisation of a series of T cell specific cDNA clones probably encoding one chain of the T cell receptor molecule (1, 2). Corresponding genomic clones have been isolated from -phage libraries prepared from liver DNA, and from the I-E restricted, cytochrome C specific T helper hybridoma 2B4. Restriction and sequence analysis of these clones indicates that the functional gene is formed by V-D-J DNA rearrangements followed by RNA splicing of the V-D-J segment to the constant region, as in immunoglobulin. Two clusters of J regions, each followed by a 4-exon constant region have been identified and sequenced. The J regions show significant homology to immunoglobulin J regions and have conserved nonamer and heptamer flanking sequences as in immunoglobulin. These are not the same as those of immunoglobulin, however. Whereas each J region is different, the two C regions are extremely similar. No amino acid differences have been found in the first (external domain) exon or in the second, hinge-like region. Two changes have been observed in the transmembrane region exon and in the cytoplasmic region. Both the J-C regions are expressed by T cells and it appears that the 5' J regions always associate with the 5' C region and the 3' J's with the 3' C region. Studies on the differential expression of these sets of genes in functional T cells are in progress. N.R.J.G. is supported by the Cancer Research Institute Inc. 1. HEDRICK, S. M., COHEN, D. I., NIELSEN, E. A., and DAVIS, M. M. 1984. Nature 308: 149. 2. HEDRICK, S. M., NIELSEN, E. A., KAVALER, J., COHEN, D. I., and DAVIS, M. M. 1984. Nature 308: 153.
Montreal General Hospital Research Institute, Montreal, Quebec, Canada
371. The significance of binding of chicken anti-immunoglobulin antibodies to human T cells D. G. HAEGERT, M. TRUDEL, and MARTINA TIMM
Two chicken anti-human FV!-l antibody preparations with strong anti-VH activity bound to approximately 20 % of normal human T cells as demonstrated by the mixed antiglobulin rosetting reaction. A third reagent, raised against a human neoplastic T cell line (CCRF-HSB2) then made immunoglobulin (Ig)-specific by purification on insolubilized human F(ab')z, reacted with approximately 90 % of T cells. T cell reactivity was shown not to be due to recognition of adsorbed Ig, carbohydrate moieties, MHC antigens or ~2 microglobulin. Antigen binding inhibition experiments and capping experiments, in which T3 molecules comodulated with determinants demonstrated by anti-CCRF-HSB-2, strongly suggest that two of the three reagents react with the T cell antigen receptor. Serological and surface marker studies and a competitive radioimmunoassay experiment using chicken anti-CCRF-HSB-2 antibodies and 1251 labelled F(ab'Jrcross-reactive materials isolated from CCRF-HSB-2 cells indicates that binding of chicken anti-Ig to T cells reflects cross-reactivity of T cell determinants with Ig and is not due to detection of Ig-gene products in T cells.
244 . 16th International Leucocyte Culture Conference, Cambridge Clinical Immunology Research Centre, University of Sydney, Sydney, Australia
372. A cytoplasmic determinant present in certain transformed or activated T cells is recognized by a monoclonal antibody to free kappa chains G. E. HAYDEN, R. L. RAISON, C. C. GOODNOW, and K. Z. WALKER
A determinant designates STA (Sezary T cell antigen) has been identified in the cytoplasm of Sezary T cells, some human T cell lines and activated T cells using a monoclonal antibody (K1-21) raised against free human kappa chains. This determinant was originally observed in the E-rosette positive (E+) cell population derived from peripheral blood (4/4 cases), lymph node or bone marrow of patients with Sezary Syndrome. These cells were of the Leu3a+, 2a- cell surface phenotype. Subsequently, STA was identified in the cytoplasm of six out of eight influenza virus specific human T cell clones of Leu3a+ phenotype, two human T-ALL cell lines, CCRF-CEM (Leu3a+, 2a-), CCRF-HSB (Leu3a-, 2a-) and the Gibbon T lymphoma line MLA-144 (Leu3a-, 2a-). E+ cells from normal donors did not exhibit the STA determinant. However, in vitro PHA stimulation of these cells for 5 days resulted in 93 % expression of STA. In all the T cells examined, STA expression was restricted to the cytoplasm and could not be detected on the cell surface. In addition, all STA + T cells failed to react with polyvalent antisera to human light chains or immunoglobulins. K-1-21 recognises a conformation dependent epitope on free kappa light chains and thus the reactivity observed in certain T cells may reflect some conformational homology between kappa light chains and the molecule carrying the STA determinant. This is of particular interest in view of recently demonstrated homologies between immunoglobulin genes and a putative gene encoding the T cell antigen receptor (1).
1. BERZOFSKY, J. 1983. Immunol. Today. 4: 299.
lThe University of California at San Diego, La Jolla, CA 92093, 2Laboratory of Immunology, NIAID, Bethesda, MD 20205, and 'Stanford University, Stanford, CA 94305, U.S.A.
373. Molecular analysis of one chain of the T cell receptor for antigen S. M. HEDRICK!, R. N. GERMAIN2, J. ROTHBARD', and M. M. DAVIS'
A chain of the T cell receptor has been extensively characterized in terms of sequence and patterns of cellular expression. A cDNA clone was obtained for a portion of the antigen receptor on T helper cells by making three assumptions: (1) The T cell receptor is expressed exclusively in T lymphocytes; (2) It is a membrane protein translated on membrane-bound polysomes; and (3) The genes undergo rearrangement in mature T lymphocytes. A comparison of the sequences of several homologous cDNA clones isolated from a thymocyte library indicate that the receptor chain is at least composed of a variable region, a joining region, and a constant region. These regions show significant sequence homologies at the amino acid level to the corresponding regions of all three immunoglobulin chains. Additionally, the amino acid sequence derived from the DNA sequence indicates the presence of leader peptide at the amino-terminus, a membrane spanning sequence, and short hydrophillic stretch of amino acids at carboxy-terminus indicative of a cytoplasmic piece. The genomic sequences that hybridize to this cDNA clone are rearranged in all hybridoma T helper cells tested (12/12), most cytotoxic T cells and T cell hybridomas (5/6), but very few T suppressor cell hybridomas (2/14).
16th International Leucocyte Culture Conference, Cambridge· 245 The rearrangements in T helper cells appear to correlate with antigen specificity, and a comparison of variable region sequences of these cells is in progress and will be discussed. Conclusive evidence that the gene we have cloned encodes a portion of the receptor was obtained by the use of rabbit antisera made to synthetic peptides predicted by the DNA sequence data. Such antisera have proven effective in blocking an antigen-specific T cell response.
Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University Medical School, Baltimore, MD, U.S.A.
374. A novel leucocyte antigen involved in the induction phase but not effector phase of T cell responses J. E. K. HILDRETH and J. T. AUGUST Recent studies have demonstrated that at least six cell surface structures are involved in T cell functions. These are the antigen receptor, the Interleukin-2 receptor, the lymphocyte functionassociated (LFA) molecule, and the T3, T4, and T8 antigens. All of these structures except LFA, which is expressed on all leucocytes, are phenotypic markers of T cells. We have produced a monoclonal antibody recognizing a new leucocyte antigen involved in T cell responses. Balb-c mice were immunized with human adherent mononuclear cells and hybridomas were generated by fusing the immune spleen cells with the P3X653.Ag8 myeloma line using polyethylene glycol. Monoclonal antibodies (MAb) secreted by hybridoma lines were screened in functional assays for inhibition of T cell responses. One MAb (H4C4) was found to completely inhibit the mixed lymphocyte reaction (MLR) and response to a soluble antigen (tetanus toxoid). The antibody had no effect on phytohemagglutinin (PHA) or pokeweed mitogen induced proliferation. The antibody also failed to inhibit natural killer cell and cytotoxic T cell (CTC) mediated lysis of target cells. Indirect radioimmunoassays and immunohistological studies indicated that the H4C4 antigen is expressed on T cells, B cells, and monocytes, but not on granulocytes. Immunoprecipitation from surface iodinated cells showed that the H4C4 antigen is a single polypeptide of 85,000 daltons. Our results show that the H4C4 antigen is a leucocyte common antigen involved in the antigenic but not mitogenic, induction of T cell responses. Studies in which cells are preincubated with antibody suggest that the H4C4 MAb inhibits at the level of the T cell, not the stimulating or antigen presenting cell. The failure of H4C4 MAb to inhibit CTC killing suggests that the H4C4 antigen is involved in the induction phase but not effector phase of T cell responses. Supported by NIH Grants POI AI 19373-02 and R01 GM 31168, and ONR N00014-82-k0221.
Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021, U.S.A.
375. Induction of suppressor cells by the OKT3/anti-Leu 4 monoclonal antibodies JOLANTA KUNICKA and C. D. PLATSOUCAS Monoclonal antibodies recognizing the T3/Leu 4 T cell differentiation antigen exhibit profound effects on various T cell functions, including inhibition of: (1) proliferative responses
246 . 16th International Leucocyte Culture Conference, Cambridge of T cells to mitogens and to allogeneic cells in mixed lymphocyte culture (MLC); (2) T cell helper function to B cell responses; (3) the effector phase of specific T-cell mediated cytotoxicity against allogeneic or autologous chemically modified targets. Furthermore, the OKT3/antiLeu 4 monoclonal antibodies are mitogenic, induce the production of gamma interferon and augment natural killer cytotoxicity. We report here that in vitro treatment of human peripheral blood mononuclear leucocytes (MNC) with OKD or anti-Leu 4 monoclonal antibodies resulted in induction of suppressor cells. OKT3/anti-Leu 4 treated cells were able to suppress significantly proliferative responses of autologous MNC to PHA in 13 of 17 normal donors. Furthermore, MNC treated with these monoclonal antibodies significantly suppressed the proliferative responses of autologous lymphocytes to allogeneic cells in MLC, in all donors examined (seven). Maximum suppression of proliferative responses was observed after treatment with the OKT3 or anti-Leu 4 monoclonal antibodies for forty eight hrs, although significant suppression was observed in certain donors after treatment for twenty four hrs only. After treatment for seventy two hrs with these monoclonal antibodies diminished suppression was observed. Proliferative responses to the OKT3/anti-Leu 4 monoclonal antibodies were observed after treatment for forty eight hrs or longer. Furthermore, immunoglobulin production by MNC in the PWM-induced differentiation system was suppressed by OKT3/anti-Leu 4 induced cells in certain donors. Treatment of mononuclear cells with monoclonal antibodies recognizing other T cell differentiation antigens (anti-Leu 2a, anti-Leu 3a) did not result in induction of suppressor cells. Studies are in progress to elucidate whether or not OKT3/anti-Leu 4-induced suppressor cells playa role in the inhibition of proliferative responses of T cells caused by these antibodies. Supported by grant CA-32070 from NIH and grant CH-151 from ACS.
Laboratory of Immunology, NIAID, NIH, Bethesda, Md 20205, U.S.A.
376. T-Iymphocyte functional antigens: The L3T4-antigen and the Ly-l antigen L. LOGDBERG, P. WASSMER, and E. M. SHEVACH In an attempt to make monoclonal antibodies (Mab) to cell surface antigens involved in T lymphocyte function, we prepared rat/mouse hybridomas against mouse thymocytes (single immunization i.v. + i.p.; fusion 4-5 days later). The hybridomas were screened for their ability to interfere with thymocyte co-stimulator (Ill) assay. Monoclonal IgM-producing hybridoma-lines were established and two of them, 2B6 (inhibition) and 2F10 (enhancing), were selected for further investigation. Mab 2B6 reacts with 90 % of thymocytes and on splenocytes the 2B6 antigen is expressed on a T lymphocyte subpopulation, mutually exclusive to the Lyt2-positive subpopulation. Thus, the 2B6 antigen appeared similar to the L3T4 antigen, as recently defined by the monoclonal antibody GK1.5: 1) 2B6 and GK1.5 gave very similar flow cytometry staining patterns on thymocytes, purified spleen T -cells and all tested T cell hybridomas. 2) Depletion of 2B6-positive cells with antibody and complement led to simultaneous depletion of GK1.5 positive cells, and vice versa. 3) Depletion of Lyt2-positive cells led to enrichment of both 2B6- and GK1.5-positive cells. 4) Both 2B6 and GK1.5 immunoprecipitate molecules of about 55,000 daltons from detergent extracts of cell surface labeled thymocytes. 5) Like GK1.5, the 2B6 antibody blocks MHC class II restricted T cell function (MLR, antigen-induced lymphocyte transformation, antigen-induced IL2-secretion from T-cell hybrids). On the other hand, 2B6 and GK1.5 apparently recognize distinct epitopes, as they do not cross-block each other. The determinant recognized by GK1.5 is called L3T4a. We suggest that the determinant recognized by 2B6 be named UT4b. The 2F10
16th International Leucocyte Culture Conference, Cambridge· 247 antibody apparently recognizes the Ly-l molecule. 1) It immunoprecipitates a 67,000 dalton molecule from detergent extracts of cell surface labeled thymocytes. 2) 2FI0 and a previously established anti-Ly-l antibody (53.7.3) give identical flow cytometry staining patterns. 3) 2FIO and 53.7.3 cross-block each other. The 2FI0 antibody markedly enhanced the response to III in the thymocyte co-stimulator assay, and also the response of thymocytes to PHA alone, thus acting much like III itself. These results raise the possibility that the Ly-l molecule may function as an Ill-receptor.
Institute of Biochemistry, University of Lausanne, Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland
377. Biosynthesis and maturation of the Lyt-2/3 antigenic complex of mouse T lymphocytes in vivo B. LUESCHER, H. R. MACDONALD, and C. BRaN
The Lyt-2I3 complex, which is selectively expressed on mouse thymocytes and cytolytic T lymphocytes is composed of three polypeptides with apparent molecular weight of 36-38,000 (a), 31-33,000 (~) and 26-28,000 (y). We have investigated the in vivo biosynthesis of this complex in thymocytes. The three chains were labelled intracellularly in pulse-labelled cells by immunoprecipitation with three different monoclonal antibodies directed against monomorphic or polymorphic determinants of the molecules. After 10 min pulse with 35S-methionine and 35S-cysteine, the three chains detected carried «high mannose» oligosaccharide units of apparent molecular weights 31-33>000 (0), 28-29,000 (~) and 22-23,000 (y). These were then processed to the complex form during subsequent transport to the cell surface. The treatment of these early forms with endo-~-N-acetylglucosaminidase F (endo-F) generated polypeptide precursors of 20-21,000 (0), 16,000 (~) and 18>500 (y) molecular weight. Endo-F treatment of Lyt-2/3 antigens expressed on the cell surface revealed that the mature 0 and ~ chains each carry three Asn-linked carbohydrate units of which one is of the «high mannose» type as indicated by its susceptibility to endo-~-N-acetylglucosaminidase H (endo-H) treatment. In contrast, the mature y chain carries only one unit of the complex type. A difference in molecular weights of 3-5,000 daltons was observed after endo-F treatment of surface expressed molecules and their «high mannose» forms. This suggests further post-translational events such as O-glycosylation during the maturation of the three chains. This is further supported by the charge heterogeneity after endo-F digestion of mature polypeptides observed by separation on two-dimensional gels.
Laboratory of Immunology, NIAID, Bethesda, MD. 20205> U.S.A.
378. The locations and total numbers of cysteines affect the conformation and expression of rabbit kappa light chains and may have a similar effect on analogous T cell receptors structures R. G. MAGE, N. MCCARTNEy-FRANCIS> E. LAMaY!> A. ANGIOLILLO> and K. E. BERNSTEIN Heterozygous rabbits of Kl allotypes b4b9 or b5b9 have equal numbers of pre-B cells expressing each allelic allotype> but mature secreting cells of b4 or b5 type predominate over
248 . 16th International Leucocyte Culture Conference, Cambridge b9. In homozygous b9 rabbits, up to 40 % of their immunoglobulins (Igs) bear Alight chains. A mutation in a b9 rabbit that was producing both Kl,b9 and K2,bas light chain isotypes led to the Basilea strain in which pre-B cells with bas or b9 are detectable but the majority of secreted Igs are of A type and small amounts of K2,bas are produced. Our nucleic acid sequences of cDNA clones encoding K2,bas from a Basilea rabbit and Kl,b9 from a normal b9 rabbit suggest that the encoded protein structures, specifically the numbers and locations of cysteines (Cys) affect their eventual expression on secreted Igs. All known rabbit Kl proteins have a Cys at position 171 in the C region that forms an interdomain disulfide bond with a Cys at position 80 in the V region, but K2,bas lacks the C region Cys. The Kl V and C genes appear to have co-envolved Cys that can form an interdomain disulfide bond. We found a K2,bas and a Kl,b9 cDNA clone that each lacked V region Cys 80. The two V. sequences were 93 % homologous and may represent a relatively small family of related V. genes. The b9 cDNA sequence that lacks Cys 80 encodes a Cys at position 108 in the J region. In three dimensional models this Cys appears capable of forming a disulfide bond with Cys 171. A second b9 cDNA clone encodes both Cys 80 and Cys 108; the translation product of this cloned mRNA would have a free SH. Similarly, chains with a free SH in the V region would result if a typical V. encoding Cys 80 is rearranged and expressed with the K2,bas isotype that lacks the C region Cys. These free SH groups might lead to non-functional or poorly functional cells or molecules. We have compared our rabbit x light chain sequences with recently published cDNA sequences that encode one chain of the human and mouse T cell receptor. Extra V, J and C region Cys in these sequences occur at positions analogous to but not identical with those found in rabbit Kllight chains. Protein and DNA match programs that maximize homologies align the intradoma in disulfide bond-forming Cys of both T cell receptor sequences to those in our x light chain sequences. Each T cell gene's C region Cys aligns at homology position 174 of the Kabat et al. numbering system for x light chains. The mouse V region Cys aligns at homology position 65. The human sequence lacks a V region Cys but has one at the beginning of a putative J-encoded region. It is likely that as with the rabbit x chains, this chain of the T cell receptor is affected in its conformation, expression and reactivity by the potential free SH groups and alternative disulfide bonds that may form depending upon the locations and total numbers of Cys residues.
Ludwig Institute for Cancer Research, Lausanne Branch, Switzerland
379. Monoclonal antibodies directed to clonotypic or common determinants involved in human T cell receptor activity A. MORETTA, S. CARREL, G. PANTALEO, and L. MORETTA
Recently we provided evidence for a clonal heterogeneity in the requirement for T3, T8 and T4 molecules in human CTL function (1). In addition, we showed that T3 molecules are not always physically associated to the antigen receptor structures on the CTL surface. The T cell receptor molecules have been so far identified by monoclonal antibodies (mAbs) recognizing clonotypic structures, while no mAbs have been described that are directed against constant portions of the molecule. In an attempt to raise mAbs specific for the T cell receptor structure, we immunized mice with the A28 MLC-derived CTL clone. The specific CTL activity of A28 clone (T3+' T8+, T4-) was resistant to inhibition by B9.4 (anti-TS) or OKT3 (anti-D) mAbs. Out of 980 individual hybridomas selected in HAT medium, 3 produced mAbs (Tcl 1-3) which reacted with the immunizing clone but failed to bind to resting or activated T cells, B cells and severallymphoblastoid or melanoma cell lines. Moreover, Tcl 1-3 inhibited CTL activity of the immunizing clone, but did not affect the specific cytolysis of MLC-activated T cell populations or T cell clones derived from the same individual or from unrelated donors.
16th International Leucocyte Culture Conference, Cambridge· 249 Taken together, these data indicate that Tcl 1-3 mAbs react with clonotypic structures of the A2S clone. In addition to Tcll-3, another mAb (Trl) was found to inhibit CTL activity of the A2S clone, however, it also blocked specific cytolysis of MLC populations and of all 52 MLCderived CTL clones analyzed (note that 32 % and 25 % of such clones were unaffected by large amounts of OKT3 or B9.4 mAbs, respectively). Natural-Killer (NK)-like and phytohemagglutinin (PHA)-dependent cytolytic activity of MLC T cells or T cell clones were unaffected by Trl mAb. Trl mAb completely inhibited cell proliferation in MLC, but had no effect on PHA- or IL-2-induced T cell growth. In addition, it was found to inhibit the alloantigeninduced IL-2 production by alloreactive helper clones. The corresponding antigen was expressed on all thymocytes and peripheral T cells (whether resting or activated) but was absent on B cells or monocytes. All together these data are consistant with the idea that Trl mAb reacts with a molecule involved in the receptor activity of all T cells. This concept was further substantiated by experiments in which the antigen recognized by Tr1 mAb was shown to cocap completely with molecules bound by Tcll-3 clonotypic mAbs. 1. MORETTA, A., G. PANTALEO, M. C. MINGARI, L. MORETTA, and J. C. CEROTTINI. 19S4. Clonal heterogeneity in the requirement for T3, T4 and TS molecules in human cytolytic T lymphocyte function. J. Exp. Med. 159: 921.
National Institutes of Health, Immunology Branch, NCI, Bethesda, Maryland, 20205, U.S.A.
380. Differential expression of class II MHC antigens on porcine helper (Th) and cytotoxic (Tc) T cell subsets M. D. PESCOVITZ, JOAN K. LUNNEY, and D. H. SACHS
Resting porcine nylon wool nonadherent cells (T cells) have previously been shown to express class II MH C antigens as determined by the reactivity of class II specific alloantisera (1). The monoclonal antibody, 40D (anti-murine I-Ek), has previously been shown to react with porcine class II antigens by immunoprecipitation (2). When this antibody was used to stain a population of porcine T cells by flow microfluorimetry (FMF) only 60 % of these cells were seen to express class II antigens. It was therefore of interest to determine whether the presence of class II antigens marked functionally distinct T cell subsets. This question has been addressed using two recently prepared monoclonal antibodies, PTS and PT4, in conjunction with in vitro cellular assays. PT4 and PTS together reacted with 90 % of nylon wool nonadherent cells and divided these T cells into distinct subsets as determined by 2 dimensional FMF analysis and antibody mediated cytotoxicity on purified peripheral blood lymphocyte populations. Treatment with PTS and complement eliminated both Tc precursors and effectors whereas treatment with PT4 and complement eliminated cells proliferating to both soluble and alloantigens but had no effect on cytotoxic T cells. This data plus the molecular weights of the precipitated antigens (35,000 for PTS and 55,000 for PT4, under reducing conditions) indicated that these monoclonal antibodies defined the porcine T c and Th subpopulations. Using these antibodies and 40D, we have examined the cellular distribution of class II antigens by 2 dimensional FMF analysis. 90 % of the PTS+ cells were found to be Ia+ whereas only 30 % of the PT4 + cells were Ia +. Similar analysis of MLR blasts showed the same pattern of Ia + cells as seen with resting T cells in that all PTS+ cells expressed Ia while only half of PT4 + cells expressed Ia. This suggests that the Ia phenotype was fixed and that expression was not dependent solely on activation. The distribution of class II antigens on functional populations was further examined by the effect of 40D and complement treatment on MLR and CTL reactivity. Treatment of the MLR responder population resulted in no decrease and sometimes an enhancement in proliferative activity. (This treatment did eliminate
250 . 16th International Leucocyte Culture Conference, Cambridge all allostimulatory capacity of the same population indicating completeness of the elimination). Despite the significant MLR proliferation, this population had a 75 % decrease in CTL activity when tested on day 7. In addition, treatment of a CTL effector population with 40D and complement eliminated the majority of CTL activity. These results indicate that class II MHC antigens are differentially expressed on porcine Th and Tc and suggest a possible functional relevance to this expression.
1. THISTLETHWAITE, J. R., Jr., L. R. PENNINGTON, J. K. LUNNEY, and D. H. SACHS. Transplantation 35: 394, 1983. 2. LUNNEY, J. K., B. A. OSBORNE, S. O. SHARROW, C. DEVAUX, M. PIERRES, and D. H. SACHS. J. Immunology 130: 2786, 1983.
Institute for Clinical Immunology, Inselspital, Bern, Switzerland, and !Department of Clinical Immunology, Medical School Hannover, Hannover, FRG
381. Characterization of different epitopes on the T8 glycoprotein W. J. PICHLER, G. WOLFF-VORBECKt,
c. H. WALKER, and H. H. PETER!
To analyze the functional role of the T8 antigen, which is present on ca. 30 % of circulating T cells, ten monoclonal antibodies directed versus the T8 glycoprotein were studied for their inhibitory activity on MLR stimulated cytotoxic T cells and with respect to enzyme sensitivity of the epitopes recognized. The ten monoclonal antibodies react with different epitopes of the same antigen as shown by identical MW of ca. 76 KD, cocapping, and blocking of antibody binding. The epitopes recognized by the antibodies differed as they differ in their sensitivity to trypsin or papain and also because two epitopes were not expressed on rhesus macacae T lymphocytes. The inhibitory activity of the monoclonal antibodies was different, as those antibodies recognizing a highly trypsin sensitive region were almost always inhibitory, while the antibodies reacting with a relatively trypsin resistant region of the T8 glycoprotein inhibited the cytotoxicity in certain effector/target cell combinations only. Thus, the combined use of enzymatic digestion, phylogenetic comparisons and blocking studies may allow to define functionally distinct regions on the T8 glycoprotein and thus help to understand the function of the T8 'glycoprotein.
Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, P.O. Box 6120, Wiirzburg, FRG
382. Effect of monoclonal antibodies against rat leucocyte differentiation antigens on activation of in vitro selected encephalitogenic T line cells H.
J. SCHLUESENER and H. WEKERLE
Lymphocyte differentiation antigens can be more than mere descriptive markers of different lymphocyte subsets. Some of them are structures with definite functions corresponding to the differentiated status of the cell. Here we describe the effect of monoclonal antibodies against
16th International Leucocyte Culture Conference, Cambridge· 251 rat lymphocyte differentiation antigens and against rat MHC products on in vitro activation of encephalitogenic T cells. A Myelin Basic Protein (MBP) specific T cell line, vLe, was selected from naive, unprimed Lewis rat lymph node cells. The line cells display the rat T cell markers W3/13 and W3125 but are negative for OX 8 and OX 22. Upon activation with MBP presented by syngeneic accessory cells, the blasts transiently express la antigens as assessed by staining with monoclonal antibodies OX 3 and OX 6, which recognise polymorphic and monomorphic determinants of rat Ia respectively. la expression of the blast is concomitant with the ability to efficiently transfer lethal Experimental Allergic Encephalomyelitis (EAE) to normal adult syngeneic recipients (LDso: 1 x 106 cells/rat). The activation process can be blocked in vitro by addition of various monoclonal antibodies reactive with the responding T line cells or with the antigen presenting cells. The monoclonal antibodies OX 3 and OX 6 block T cell line activation at ascites dilutions of 1 :50,000, while antibody OX 17, reactive with a monomorphic determinant on the rat 1-E equivalent, is ineffective. This finding could suggest that responses against MBP in the Lewis rat are restricted solely by I-A region like gene products of the rat RT1.B complex. Furthermore, activation of T lymphoblasts could be blocked by antibodies directed against T cell differentiation antigens such as W3125, a putative analog of the human T4 marker.
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
383. The induction of non specific cytotoxic activity in human T-cell clones by antibodies against T3 H. SPITS, H. YSSEL, and E. DE VRIES The 13 antigen is a complex of at least 5 different proteins which is expressed on mature T cells. This complex appears to be involved in T-cell function. The activation of T cells by antigen can be mimicked by monoclonal anti-T3 antibodies. This is understandable since recent findings indicate that the receptor for alloantigen is associated with T3. Here we report that anti-T3 reagents, in addition to their capacity to induce proliferation and to block antigen specific cytotoxic activity mediated by cytotoxic T-cell (CTL) clones, induce non specific cytotoxic activity in cytotoxic T-cell clones. Furthermore we found that anti-T3 reagents were able to trigger cytolytic activity in T-cell clones, which were characterized as non cytotoxic antigen specific proliferating T cells. These results indicate that non cytotoxic T helper cells may also have a lytic machinery which can be triggered by anti-T3 reagents.
Laboratoire d'lmmunogenetique Humaine, C.H.U. Pitie-Salpetriere, 91, boulevard de I'Hopital, 75013 Paris, France
384. Biochemical evidence for clonotypic differences in antigen specific T cell clones T. TRIEBEL, P. DEBRE, and D.]. CHARRON Biochemical analysis of antigen specific T cell clones from a single individual may reveal clonotypic differences which could help to define new surface determinants such as differentia-
252 . 16th International Leucocyte Culture Conference, Cambridge tion antigens or antigen receptors. We obtained varidase (Var) or diphtheria toxoid (DT) specific cloned T cells from a single individual DR 6/7. These cloned cells were homogeneous in terms of membrane markers (OKT3+, OKT4+, OKTS-, RFB-l+, DR+) at a given stage of activation (3 days after stimulation with IL2). NP 40 totallysates of 35S methionine labelled cloned T cells were analysed by 2D-PAGE (IEF was used in the first dimension and a 10% SDS-PAGE in the second dimension). Three different spots were considered as present or missing in some of the clones tested after repeated electrophoresis of the same cell lysate. Spot 1 is very acidic with a MW of approximately 150 Kd. Spot 2 is slightly less acidic with a MW of 120 Kd. Spot 3 is very basic with a MW of 36 Kd. This biochemical heterogeneity demonstrate clonotypic differences in antigen specific T cell clones. The experimental approach described here may help to define important cellular proteins which could possibly reflect a given cellular function, specificity or differentiation stage.
Rotterdam Radio-Therapeutic Institute, The Netherlands
385. Human cytotoxic T cell clones show differential susceptability for
enhancement and/or inhibition of nonspecific cytolysis by lectins and anti-T3-MCA
R.
J. VAN DE GRIEND, C.
P. M. RONTELTAP, and R. L. H. BOLHUIS
The role of cell surface determinants as for instance recognized by the monoclonal antibodies (MCA) T3, T4, T8 etc. on the cytolytic activity of cytotoxic lymphocytes appears to be a complex one. In some T3+ T8+ clones we found that the HLA specific cytolytic activity can be blocked by OKT8 and by OKT3 (partially) MCAs, but that the cytolytic activity of other T3+ T8+ clones is not reduced at all. Two clones, Dll and G9, did not show cytolytic activity against Daudi cells but showed cytolytic activity against K562. However, when incubated with high concentrations of MCA against the T3 receptor, but not the T8 receptor, non-specific cytolysis against Daudi cells can be induced and the lytic activity against K562 augmented. Similar effects were obtained by preincubation of the effector cells with PHA or leucoagglutinin. In contrast, no cytolytic activity against Daudi cells was induced MCA in an T3+T4+ (n+20) allospecific cytotoxic clone, by OKT3 or OKT4 whereas those antibodies (partially) inhibited the cytolytic activity of this clone against the original stimulator cell. PHA had only a marginal effect on the cytolytic activity of this clone. Furthermore the cytolytic activity of OKT3-T4-T8-Tl1+ activated killer clones (recently established by us) is not augmented or inhibited by OKT3 or by lectins. The effects of lectin and OKT3 on the cytolytic activity of T-cell clones does apparently not depend on the presence or absence on the T3 marker but can also be different for T3+ clones varying from no effect on OKT3clones, an inhibitory effect on the anti class II T3+ T4+ clone (n+20), enhancement and/or induction of lytic activity on T3+ T8+ clone Dll or both inhibition and enhancement on T3+ T8+ clone G9. The cytotoxicity promoting activity of OKT3 on these clones thus resembles the effect of lectins, although quantitative differences exist. When compared with lectin, OKT3 has a stronger effect on the cytolytic activity against Daudi cells whereas the lysis of K562 is more augmented by PHA.
16th International Leucocyte Culture Conference, Cambridge . 253 Laboratory of Immunology, NIAID, NIH, Bethesda, Md 20205, U.S.A.
386. Inhibition ofT cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells P. WASSMER, C. CHAN, L. LOGDBERG, and E. M. SHEVACH
Two monoclonal antibodies (Mab) to distinct epitopes of the UT4 antigen (Mab, GK1.5 and 2B6) were tested for their capacity to block IL-2 secretion by concanavalin A (Con A) and antigen activated T-cell hybridomas. The T-cell hybridomas were MHC class II-restricted, insulin or ovalbumin specific, and of BALB/c or C57BL/6 origin. The induction of IL-2 production by antigen and syngeneic accessory cells (AC) was markedly susceptible to inhibition by Mab GK1.5. In general, 2B6 produced less inhibition of antigen-induced T cell activation. High concentrations of antigen could partially overcome the antibody induced inhibition. This result suggests a possible relationship between the susceptibility to blocking and the strength of the activation signal. We next examined the effect of anti-UT4 Mab on the ConA induced activation of the same panel of T cell hybridomas in an AC free culture. We found that both Mab were capable of producing marked inhibition of ConA induced IL-2 secretion although the susceptibility of the different hybridomas to blocking was more variable than that seen in the response to antigen. Again, an excess of the activation signal (higher concentrations of ConAl could partially overcome the inhibitory effect. In contrast to antigen driven IL-2 production, 2B6 was more potent than GK1.5 in blocking ConA induced activation. Because T-cell hybridomas were found to be Ia antigen negative as d_etermined by FACS analysis, the blocking of ConA activation could be demonstrated in a presumably MHC class II antigen free assay system. This observation is not consistent with the hypothesis that anti-UT4 antibodies inhibit T cell activation by interfering with interactions between UT4 and MHC class II products. An alternative explanation which is suggested by our findings is that the UT4 antigen interacts with the T cell receptor and plays a role in transmitting the activation signal as part of a multi-molecular complex. We cannot, however, exclude that anti-UT4 antibodies may inhibit T cell activation by more than one mechanism.
I.C.R.F. Human Tumour Immunology Group and Department of Clinical Haematology, School of Medicine, University College London, London WC1, U.K.
366. Human T lymphocyte activation investigated using mitogenic monoclonal antibodies P. C. L. BEVERLEY, D. WALLACE, E. D. ZANDERS, K. O'FLYNN, and D. LINCH
Antibodies to the T3-receptor complex of human T lymphocytes may be mitogenic. While the IgG2a anti-T3 antibody OKT3 stimulates peripheral blood mononuclear cells (PBM) of all normal donors, the IgG1 antibodies UCHTl and Leu 4 stimulate PBM from only 70 % of individuals. The response of non-responders can be reconstituted with accessory cells from responders (1). This system provides a model for investigation of accessory cell-T cell interaction. We have used the calcium sensitive dye Quin-2 to investigate the effects of antibodies to the T3-receptor complex and have shown that UCHTl can trigger a calcium flux in both responder and non-responder lymphocytes confirming both that non-responder T cells react «normally» to ligand binding to T3 and that a second signal provided by accessory cells is required for initiation of DNA synthesis in T cells. In further experiments we have attempted to define what is provided by the accessory cells using agents which crosslink the anti-T3 antibody or stimulate or replace the accessory cells. In addition we have attempted to define to what extent the method of accessory cell activation regulates the subset of T cells activated or the pathway of differentiation of the activated cells (2). 1. TAX, W. J. M., WILLIAMS, H. W., REEKERS, P. P. M., CAPEL, P. J. A., and KOENE, R. A. P. Polymorphism in mitogenic effect of IgG1 monoclonal antibodies against T3 antigen on human T cells. Nature 304: 445--447 (1983). 2. VAN WAUWE, J. P., GOOSSENS, J. G., and BEVERLEY, P. C. L. Human T lymphocyte activation by monoclonal antibodies: OKT3, but not UCHTl, triggers mitogenesis via an interleukin 2-dependent mechanism. J. Immunol. in press (1984).
Department of Experimental Pathology, St. Mary's Hospital Medical School, London, U.K.
367. Demonstration of a third component of the human T cell antigen complex A. W. BOYLSTON, R. D. GOLDIN, and C. S. MOORE HPB-All is a human T cell leukaemia line which expresses a clonotypic molecule with all the properties of the putative T cell antigen receptor, i.e., comodulation with the T3 antigen, heterodimeric structure with. 50,000 and 40,000 molecular weight subunits; and strict
242 . 16th International Leucocyte Culture Conference, Cambridge clonotypy. Whilst investigating other McAbs raised against HPB-All a McAb (A5) with the ability to comodulate the TCLIi antigen of HPB was discovered which has a structure different from TCLIi and T3 but which has properties suggesting that it is also part of the antigen receptor complex. These are: 1) A5 comodulates T3 and TCLIi on HPB-All, 2) A5 comodulates T3 on normal T cells, 3) A5 is mitogenic for normal T lymphocytes, 4) A5 has the same cell type and cell line distribution as T3, 5) A5 recognizes a molecule different from T3 or TCLIi because it precipitates a membrane component with a MW of 64,000 in SDS-PAGE under both reducing and non-reducing conditions.
Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London, WC ItE, 6BT, U.K.
368. Studies on the human T cell receptor for antigen M. K. COLLINS and M. J. OWEN Recently a mouse eDNA clone, 86Tt, which codes for a T-lymphocyte specific molecule with an immunoglobulin like structure was isolated. We have used this clone as a probe to investigate the structure of homologous molecules in human T-lymphocytes. In Northern blot analysis 86Tl detected the presence of an analogous mRNA in various human T cell lines and in functional T helper cell clones, but not in any other cell type. 86Tt was then used to isolate a eDNA clone from a library constructed in )"GTto from the T4 positive human T cell line J6. The structure of this clone will be discussed.
Department of Immunology, University of Ulm, FRG
369. Involvement of the T3 antigen in polyclonal T cell activation by mitogenic lectins and oxidation B. FLEISCHER Accessory cell (AC)-dependent mitogen-induced polyclonal T cell activation has often been used as a model for antigen-specific triggering, the precise mechanisms, however, by which T cells are triggered by mitogens, are still a matter of debate. We have recently shown that mitogenic lectins or the introduction of aldehydes into cell surface glycoproteins by oxidation activate T cells by crosslinking T cell membrane proteins with the AC surface (1). In the present study, the effect of monoclonal antibodies against the T cell surface antigens T3 and T4 on AC-dependent mitogen-induced proliferation of human 14+ T cell clones and purified peripheral blood T cells was studied. To avoid the Fe-receptor dependent mitogenic effect of the OKT3 antibodies, monocytes were replaced by Fe-receptor negative tumor cells as AC. Monoclonal antibody OKT3 but not OKT4 inhibited the response of both T lymphocyte clones and purified T cells to mitogenic lectins and oxidation. The inhibition was not due to nonspecific effects of the monoclonal antibody since it affected only the initial triggering but not the proliferation of activated T cells and it could be overcome by higher concentrations of lectin. Furthermore, OKT3 antibody at the same concentration that was inhibitory in this system was mitogenic in the presence of Fe-receptor bearing monocytes. Surface modulation of T3 but not T4 antigen led to unresponsiveness to a mitogen pulse given directly after modulation. These findings suggest that antigen-specific and mitogen-induced T cell triggering are due to the interaction with the same receptors of the T lymphocyte. 1. FLEISCHER, B. 1983. Eur. J. Immunol. 13: 970.
16th International Leucocyte Culture Conference, Cambridge· 243 Stanford University School of Medicine, Stanford, CA 94305, U.S.A.
370. Sequence Organisation of a T cell receptor gene N. R. J. GASCOIGNE, Y.-S. CHIEN, D. M. BECKER, NADINE E. LEE, J. KAVALAR, and M. M. DAVIS
We have recently described the isolation and characterisation of a series of T cell specific cDNA clones probably encoding one chain of the T cell receptor molecule (1, 2). Corresponding genomic clones have been isolated from -phage libraries prepared from liver DNA, and from the I-E restricted, cytochrome C specific T helper hybridoma 2B4. Restriction and sequence analysis of these clones indicates that the functional gene is formed by V-D-J DNA rearrangements followed by RNA splicing of the V-D-J segment to the constant region, as in immunoglobulin. Two clusters of J regions, each followed by a 4-exon constant region have been identified and sequenced. The J regions show significant homology to immunoglobulin J regions and have conserved nonamer and heptamer flanking sequences as in immunoglobulin. These are not the same as those of immunoglobulin, however. Whereas each J region is different, the two C regions are extremely similar. No amino acid differences have been found in the first (external domain) exon or in the second, hinge-like region. Two changes have been observed in the transmembrane region exon and in the cytoplasmic region. Both the J-C regions are expressed by T cells and it appears that the 5' J regions always associate with the 5' C region and the 3' J's with the 3' C region. Studies on the differential expression of these sets of genes in functional T cells are in progress. N.R.J.G. is supported by the Cancer Research Institute Inc. 1. HEDRICK, S. M., COHEN, D. I., NIELSEN, E. A., and DAVIS, M. M. 1984. Nature 308: 149. 2. HEDRICK, S. M., NIELSEN, E. A., KAVALER, J., COHEN, D. I., and DAVIS, M. M. 1984. Nature 308: 153.
Montreal General Hospital Research Institute, Montreal, Quebec, Canada
371. The significance of binding of chicken anti-immunoglobulin antibodies to human T cells D. G. HAEGERT, M. TRUDEL, and MARTINA TIMM
Two chicken anti-human FV!-l antibody preparations with strong anti-VH activity bound to approximately 20 % of normal human T cells as demonstrated by the mixed antiglobulin rosetting reaction. A third reagent, raised against a human neoplastic T cell line (CCRF-HSB2) then made immunoglobulin (Ig)-specific by purification on insolubilized human F(ab')z, reacted with approximately 90 % of T cells. T cell reactivity was shown not to be due to recognition of adsorbed Ig, carbohydrate moieties, MHC antigens or ~2 microglobulin. Antigen binding inhibition experiments and capping experiments, in which T3 molecules comodulated with determinants demonstrated by anti-CCRF-HSB-2, strongly suggest that two of the three reagents react with the T cell antigen receptor. Serological and surface marker studies and a competitive radioimmunoassay experiment using chicken anti-CCRF-HSB-2 antibodies and 1251 labelled F(ab'Jrcross-reactive materials isolated from CCRF-HSB-2 cells indicates that binding of chicken anti-Ig to T cells reflects cross-reactivity of T cell determinants with Ig and is not due to detection of Ig-gene products in T cells.
244 . 16th International Leucocyte Culture Conference, Cambridge Clinical Immunology Research Centre, University of Sydney, Sydney, Australia
372. A cytoplasmic determinant present in certain transformed or activated T cells is recognized by a monoclonal antibody to free kappa chains G. E. HAYDEN, R. L. RAISON, C. C. GOODNOW, and K. Z. WALKER
A determinant designates STA (Sezary T cell antigen) has been identified in the cytoplasm of Sezary T cells, some human T cell lines and activated T cells using a monoclonal antibody (K1-21) raised against free human kappa chains. This determinant was originally observed in the E-rosette positive (E+) cell population derived from peripheral blood (4/4 cases), lymph node or bone marrow of patients with Sezary Syndrome. These cells were of the Leu3a+, 2a- cell surface phenotype. Subsequently, STA was identified in the cytoplasm of six out of eight influenza virus specific human T cell clones of Leu3a+ phenotype, two human T-ALL cell lines, CCRF-CEM (Leu3a+, 2a-), CCRF-HSB (Leu3a-, 2a-) and the Gibbon T lymphoma line MLA-144 (Leu3a-, 2a-). E+ cells from normal donors did not exhibit the STA determinant. However, in vitro PHA stimulation of these cells for 5 days resulted in 93 % expression of STA. In all the T cells examined, STA expression was restricted to the cytoplasm and could not be detected on the cell surface. In addition, all STA + T cells failed to react with polyvalent antisera to human light chains or immunoglobulins. K-1-21 recognises a conformation dependent epitope on free kappa light chains and thus the reactivity observed in certain T cells may reflect some conformational homology between kappa light chains and the molecule carrying the STA determinant. This is of particular interest in view of recently demonstrated homologies between immunoglobulin genes and a putative gene encoding the T cell antigen receptor (1).
1. BERZOFSKY, J. 1983. Immunol. Today. 4: 299.
lThe University of California at San Diego, La Jolla, CA 92093, 2Laboratory of Immunology, NIAID, Bethesda, MD 20205, and 'Stanford University, Stanford, CA 94305, U.S.A.
373. Molecular analysis of one chain of the T cell receptor for antigen S. M. HEDRICK!, R. N. GERMAIN2, J. ROTHBARD', and M. M. DAVIS'
A chain of the T cell receptor has been extensively characterized in terms of sequence and patterns of cellular expression. A cDNA clone was obtained for a portion of the antigen receptor on T helper cells by making three assumptions: (1) The T cell receptor is expressed exclusively in T lymphocytes; (2) It is a membrane protein translated on membrane-bound polysomes; and (3) The genes undergo rearrangement in mature T lymphocytes. A comparison of the sequences of several homologous cDNA clones isolated from a thymocyte library indicate that the receptor chain is at least composed of a variable region, a joining region, and a constant region. These regions show significant sequence homologies at the amino acid level to the corresponding regions of all three immunoglobulin chains. Additionally, the amino acid sequence derived from the DNA sequence indicates the presence of leader peptide at the amino-terminus, a membrane spanning sequence, and short hydrophillic stretch of amino acids at carboxy-terminus indicative of a cytoplasmic piece. The genomic sequences that hybridize to this cDNA clone are rearranged in all hybridoma T helper cells tested (12/12), most cytotoxic T cells and T cell hybridomas (5/6), but very few T suppressor cell hybridomas (2/14).
16th International Leucocyte Culture Conference, Cambridge· 245 The rearrangements in T helper cells appear to correlate with antigen specificity, and a comparison of variable region sequences of these cells is in progress and will be discussed. Conclusive evidence that the gene we have cloned encodes a portion of the receptor was obtained by the use of rabbit antisera made to synthetic peptides predicted by the DNA sequence data. Such antisera have proven effective in blocking an antigen-specific T cell response.
Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University Medical School, Baltimore, MD, U.S.A.
374. A novel leucocyte antigen involved in the induction phase but not effector phase of T cell responses J. E. K. HILDRETH and J. T. AUGUST Recent studies have demonstrated that at least six cell surface structures are involved in T cell functions. These are the antigen receptor, the Interleukin-2 receptor, the lymphocyte functionassociated (LFA) molecule, and the T3, T4, and T8 antigens. All of these structures except LFA, which is expressed on all leucocytes, are phenotypic markers of T cells. We have produced a monoclonal antibody recognizing a new leucocyte antigen involved in T cell responses. Balb-c mice were immunized with human adherent mononuclear cells and hybridomas were generated by fusing the immune spleen cells with the P3X653.Ag8 myeloma line using polyethylene glycol. Monoclonal antibodies (MAb) secreted by hybridoma lines were screened in functional assays for inhibition of T cell responses. One MAb (H4C4) was found to completely inhibit the mixed lymphocyte reaction (MLR) and response to a soluble antigen (tetanus toxoid). The antibody had no effect on phytohemagglutinin (PHA) or pokeweed mitogen induced proliferation. The antibody also failed to inhibit natural killer cell and cytotoxic T cell (CTC) mediated lysis of target cells. Indirect radioimmunoassays and immunohistological studies indicated that the H4C4 antigen is expressed on T cells, B cells, and monocytes, but not on granulocytes. Immunoprecipitation from surface iodinated cells showed that the H4C4 antigen is a single polypeptide of 85,000 daltons. Our results show that the H4C4 antigen is a leucocyte common antigen involved in the antigenic but not mitogenic, induction of T cell responses. Studies in which cells are preincubated with antibody suggest that the H4C4 MAb inhibits at the level of the T cell, not the stimulating or antigen presenting cell. The failure of H4C4 MAb to inhibit CTC killing suggests that the H4C4 antigen is involved in the induction phase but not effector phase of T cell responses. Supported by NIH Grants POI AI 19373-02 and R01 GM 31168, and ONR N00014-82-k0221.
Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021, U.S.A.
375. Induction of suppressor cells by the OKT3/anti-Leu 4 monoclonal antibodies JOLANTA KUNICKA and C. D. PLATSOUCAS Monoclonal antibodies recognizing the T3/Leu 4 T cell differentiation antigen exhibit profound effects on various T cell functions, including inhibition of: (1) proliferative responses
246 . 16th International Leucocyte Culture Conference, Cambridge of T cells to mitogens and to allogeneic cells in mixed lymphocyte culture (MLC); (2) T cell helper function to B cell responses; (3) the effector phase of specific T-cell mediated cytotoxicity against allogeneic or autologous chemically modified targets. Furthermore, the OKT3/antiLeu 4 monoclonal antibodies are mitogenic, induce the production of gamma interferon and augment natural killer cytotoxicity. We report here that in vitro treatment of human peripheral blood mononuclear leucocytes (MNC) with OKD or anti-Leu 4 monoclonal antibodies resulted in induction of suppressor cells. OKT3/anti-Leu 4 treated cells were able to suppress significantly proliferative responses of autologous MNC to PHA in 13 of 17 normal donors. Furthermore, MNC treated with these monoclonal antibodies significantly suppressed the proliferative responses of autologous lymphocytes to allogeneic cells in MLC, in all donors examined (seven). Maximum suppression of proliferative responses was observed after treatment with the OKT3 or anti-Leu 4 monoclonal antibodies for forty eight hrs, although significant suppression was observed in certain donors after treatment for twenty four hrs only. After treatment for seventy two hrs with these monoclonal antibodies diminished suppression was observed. Proliferative responses to the OKT3/anti-Leu 4 monoclonal antibodies were observed after treatment for forty eight hrs or longer. Furthermore, immunoglobulin production by MNC in the PWM-induced differentiation system was suppressed by OKT3/anti-Leu 4 induced cells in certain donors. Treatment of mononuclear cells with monoclonal antibodies recognizing other T cell differentiation antigens (anti-Leu 2a, anti-Leu 3a) did not result in induction of suppressor cells. Studies are in progress to elucidate whether or not OKT3/anti-Leu 4-induced suppressor cells playa role in the inhibition of proliferative responses of T cells caused by these antibodies. Supported by grant CA-32070 from NIH and grant CH-151 from ACS.
Laboratory of Immunology, NIAID, NIH, Bethesda, Md 20205, U.S.A.
376. T-Iymphocyte functional antigens: The L3T4-antigen and the Ly-l antigen L. LOGDBERG, P. WASSMER, and E. M. SHEVACH In an attempt to make monoclonal antibodies (Mab) to cell surface antigens involved in T lymphocyte function, we prepared rat/mouse hybridomas against mouse thymocytes (single immunization i.v. + i.p.; fusion 4-5 days later). The hybridomas were screened for their ability to interfere with thymocyte co-stimulator (Ill) assay. Monoclonal IgM-producing hybridoma-lines were established and two of them, 2B6 (inhibition) and 2F10 (enhancing), were selected for further investigation. Mab 2B6 reacts with 90 % of thymocytes and on splenocytes the 2B6 antigen is expressed on a T lymphocyte subpopulation, mutually exclusive to the Lyt2-positive subpopulation. Thus, the 2B6 antigen appeared similar to the L3T4 antigen, as recently defined by the monoclonal antibody GK1.5: 1) 2B6 and GK1.5 gave very similar flow cytometry staining patterns on thymocytes, purified spleen T -cells and all tested T cell hybridomas. 2) Depletion of 2B6-positive cells with antibody and complement led to simultaneous depletion of GK1.5 positive cells, and vice versa. 3) Depletion of Lyt2-positive cells led to enrichment of both 2B6- and GK1.5-positive cells. 4) Both 2B6 and GK1.5 immunoprecipitate molecules of about 55,000 daltons from detergent extracts of cell surface labeled thymocytes. 5) Like GK1.5, the 2B6 antibody blocks MHC class II restricted T cell function (MLR, antigen-induced lymphocyte transformation, antigen-induced IL2-secretion from T-cell hybrids). On the other hand, 2B6 and GK1.5 apparently recognize distinct epitopes, as they do not cross-block each other. The determinant recognized by GK1.5 is called L3T4a. We suggest that the determinant recognized by 2B6 be named UT4b. The 2F10
16th International Leucocyte Culture Conference, Cambridge· 247 antibody apparently recognizes the Ly-l molecule. 1) It immunoprecipitates a 67,000 dalton molecule from detergent extracts of cell surface labeled thymocytes. 2) 2FI0 and a previously established anti-Ly-l antibody (53.7.3) give identical flow cytometry staining patterns. 3) 2FIO and 53.7.3 cross-block each other. The 2FI0 antibody markedly enhanced the response to III in the thymocyte co-stimulator assay, and also the response of thymocytes to PHA alone, thus acting much like III itself. These results raise the possibility that the Ly-l molecule may function as an Ill-receptor.
Institute of Biochemistry, University of Lausanne, Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland
377. Biosynthesis and maturation of the Lyt-2/3 antigenic complex of mouse T lymphocytes in vivo B. LUESCHER, H. R. MACDONALD, and C. BRaN
The Lyt-2I3 complex, which is selectively expressed on mouse thymocytes and cytolytic T lymphocytes is composed of three polypeptides with apparent molecular weight of 36-38,000 (a), 31-33,000 (~) and 26-28,000 (y). We have investigated the in vivo biosynthesis of this complex in thymocytes. The three chains were labelled intracellularly in pulse-labelled cells by immunoprecipitation with three different monoclonal antibodies directed against monomorphic or polymorphic determinants of the molecules. After 10 min pulse with 35S-methionine and 35S-cysteine, the three chains detected carried «high mannose» oligosaccharide units of apparent molecular weights 31-33>000 (0), 28-29,000 (~) and 22-23,000 (y). These were then processed to the complex form during subsequent transport to the cell surface. The treatment of these early forms with endo-~-N-acetylglucosaminidase F (endo-F) generated polypeptide precursors of 20-21,000 (0), 16,000 (~) and 18>500 (y) molecular weight. Endo-F treatment of Lyt-2/3 antigens expressed on the cell surface revealed that the mature 0 and ~ chains each carry three Asn-linked carbohydrate units of which one is of the «high mannose» type as indicated by its susceptibility to endo-~-N-acetylglucosaminidase H (endo-H) treatment. In contrast, the mature y chain carries only one unit of the complex type. A difference in molecular weights of 3-5,000 daltons was observed after endo-F treatment of surface expressed molecules and their «high mannose» forms. This suggests further post-translational events such as O-glycosylation during the maturation of the three chains. This is further supported by the charge heterogeneity after endo-F digestion of mature polypeptides observed by separation on two-dimensional gels.
Laboratory of Immunology, NIAID, Bethesda, MD. 20205> U.S.A.
378. The locations and total numbers of cysteines affect the conformation and expression of rabbit kappa light chains and may have a similar effect on analogous T cell receptors structures R. G. MAGE, N. MCCARTNEy-FRANCIS> E. LAMaY!> A. ANGIOLILLO> and K. E. BERNSTEIN Heterozygous rabbits of Kl allotypes b4b9 or b5b9 have equal numbers of pre-B cells expressing each allelic allotype> but mature secreting cells of b4 or b5 type predominate over
248 . 16th International Leucocyte Culture Conference, Cambridge b9. In homozygous b9 rabbits, up to 40 % of their immunoglobulins (Igs) bear Alight chains. A mutation in a b9 rabbit that was producing both Kl,b9 and K2,bas light chain isotypes led to the Basilea strain in which pre-B cells with bas or b9 are detectable but the majority of secreted Igs are of A type and small amounts of K2,bas are produced. Our nucleic acid sequences of cDNA clones encoding K2,bas from a Basilea rabbit and Kl,b9 from a normal b9 rabbit suggest that the encoded protein structures, specifically the numbers and locations of cysteines (Cys) affect their eventual expression on secreted Igs. All known rabbit Kl proteins have a Cys at position 171 in the C region that forms an interdomain disulfide bond with a Cys at position 80 in the V region, but K2,bas lacks the C region Cys. The Kl V and C genes appear to have co-envolved Cys that can form an interdomain disulfide bond. We found a K2,bas and a Kl,b9 cDNA clone that each lacked V region Cys 80. The two V. sequences were 93 % homologous and may represent a relatively small family of related V. genes. The b9 cDNA sequence that lacks Cys 80 encodes a Cys at position 108 in the J region. In three dimensional models this Cys appears capable of forming a disulfide bond with Cys 171. A second b9 cDNA clone encodes both Cys 80 and Cys 108; the translation product of this cloned mRNA would have a free SH. Similarly, chains with a free SH in the V region would result if a typical V. encoding Cys 80 is rearranged and expressed with the K2,bas isotype that lacks the C region Cys. These free SH groups might lead to non-functional or poorly functional cells or molecules. We have compared our rabbit x light chain sequences with recently published cDNA sequences that encode one chain of the human and mouse T cell receptor. Extra V, J and C region Cys in these sequences occur at positions analogous to but not identical with those found in rabbit Kllight chains. Protein and DNA match programs that maximize homologies align the intradoma in disulfide bond-forming Cys of both T cell receptor sequences to those in our x light chain sequences. Each T cell gene's C region Cys aligns at homology position 174 of the Kabat et al. numbering system for x light chains. The mouse V region Cys aligns at homology position 65. The human sequence lacks a V region Cys but has one at the beginning of a putative J-encoded region. It is likely that as with the rabbit x chains, this chain of the T cell receptor is affected in its conformation, expression and reactivity by the potential free SH groups and alternative disulfide bonds that may form depending upon the locations and total numbers of Cys residues.
Ludwig Institute for Cancer Research, Lausanne Branch, Switzerland
379. Monoclonal antibodies directed to clonotypic or common determinants involved in human T cell receptor activity A. MORETTA, S. CARREL, G. PANTALEO, and L. MORETTA
Recently we provided evidence for a clonal heterogeneity in the requirement for T3, T8 and T4 molecules in human CTL function (1). In addition, we showed that T3 molecules are not always physically associated to the antigen receptor structures on the CTL surface. The T cell receptor molecules have been so far identified by monoclonal antibodies (mAbs) recognizing clonotypic structures, while no mAbs have been described that are directed against constant portions of the molecule. In an attempt to raise mAbs specific for the T cell receptor structure, we immunized mice with the A28 MLC-derived CTL clone. The specific CTL activity of A28 clone (T3+' T8+, T4-) was resistant to inhibition by B9.4 (anti-TS) or OKT3 (anti-D) mAbs. Out of 980 individual hybridomas selected in HAT medium, 3 produced mAbs (Tcl 1-3) which reacted with the immunizing clone but failed to bind to resting or activated T cells, B cells and severallymphoblastoid or melanoma cell lines. Moreover, Tcl 1-3 inhibited CTL activity of the immunizing clone, but did not affect the specific cytolysis of MLC-activated T cell populations or T cell clones derived from the same individual or from unrelated donors.
16th International Leucocyte Culture Conference, Cambridge· 249 Taken together, these data indicate that Tcl 1-3 mAbs react with clonotypic structures of the A2S clone. In addition to Tcll-3, another mAb (Trl) was found to inhibit CTL activity of the A2S clone, however, it also blocked specific cytolysis of MLC populations and of all 52 MLCderived CTL clones analyzed (note that 32 % and 25 % of such clones were unaffected by large amounts of OKT3 or B9.4 mAbs, respectively). Natural-Killer (NK)-like and phytohemagglutinin (PHA)-dependent cytolytic activity of MLC T cells or T cell clones were unaffected by Trl mAb. Trl mAb completely inhibited cell proliferation in MLC, but had no effect on PHA- or IL-2-induced T cell growth. In addition, it was found to inhibit the alloantigeninduced IL-2 production by alloreactive helper clones. The corresponding antigen was expressed on all thymocytes and peripheral T cells (whether resting or activated) but was absent on B cells or monocytes. All together these data are consistant with the idea that Trl mAb reacts with a molecule involved in the receptor activity of all T cells. This concept was further substantiated by experiments in which the antigen recognized by Tr1 mAb was shown to cocap completely with molecules bound by Tcll-3 clonotypic mAbs. 1. MORETTA, A., G. PANTALEO, M. C. MINGARI, L. MORETTA, and J. C. CEROTTINI. 19S4. Clonal heterogeneity in the requirement for T3, T4 and TS molecules in human cytolytic T lymphocyte function. J. Exp. Med. 159: 921.
National Institutes of Health, Immunology Branch, NCI, Bethesda, Maryland, 20205, U.S.A.
380. Differential expression of class II MHC antigens on porcine helper (Th) and cytotoxic (Tc) T cell subsets M. D. PESCOVITZ, JOAN K. LUNNEY, and D. H. SACHS
Resting porcine nylon wool nonadherent cells (T cells) have previously been shown to express class II MH C antigens as determined by the reactivity of class II specific alloantisera (1). The monoclonal antibody, 40D (anti-murine I-Ek), has previously been shown to react with porcine class II antigens by immunoprecipitation (2). When this antibody was used to stain a population of porcine T cells by flow microfluorimetry (FMF) only 60 % of these cells were seen to express class II antigens. It was therefore of interest to determine whether the presence of class II antigens marked functionally distinct T cell subsets. This question has been addressed using two recently prepared monoclonal antibodies, PTS and PT4, in conjunction with in vitro cellular assays. PT4 and PTS together reacted with 90 % of nylon wool nonadherent cells and divided these T cells into distinct subsets as determined by 2 dimensional FMF analysis and antibody mediated cytotoxicity on purified peripheral blood lymphocyte populations. Treatment with PTS and complement eliminated both Tc precursors and effectors whereas treatment with PT4 and complement eliminated cells proliferating to both soluble and alloantigens but had no effect on cytotoxic T cells. This data plus the molecular weights of the precipitated antigens (35,000 for PTS and 55,000 for PT4, under reducing conditions) indicated that these monoclonal antibodies defined the porcine T c and Th subpopulations. Using these antibodies and 40D, we have examined the cellular distribution of class II antigens by 2 dimensional FMF analysis. 90 % of the PTS+ cells were found to be Ia+ whereas only 30 % of the PT4 + cells were Ia +. Similar analysis of MLR blasts showed the same pattern of Ia + cells as seen with resting T cells in that all PTS+ cells expressed Ia while only half of PT4 + cells expressed Ia. This suggests that the Ia phenotype was fixed and that expression was not dependent solely on activation. The distribution of class II antigens on functional populations was further examined by the effect of 40D and complement treatment on MLR and CTL reactivity. Treatment of the MLR responder population resulted in no decrease and sometimes an enhancement in proliferative activity. (This treatment did eliminate
250 . 16th International Leucocyte Culture Conference, Cambridge all allostimulatory capacity of the same population indicating completeness of the elimination). Despite the significant MLR proliferation, this population had a 75 % decrease in CTL activity when tested on day 7. In addition, treatment of a CTL effector population with 40D and complement eliminated the majority of CTL activity. These results indicate that class II MHC antigens are differentially expressed on porcine Th and Tc and suggest a possible functional relevance to this expression.
1. THISTLETHWAITE, J. R., Jr., L. R. PENNINGTON, J. K. LUNNEY, and D. H. SACHS. Transplantation 35: 394, 1983. 2. LUNNEY, J. K., B. A. OSBORNE, S. O. SHARROW, C. DEVAUX, M. PIERRES, and D. H. SACHS. J. Immunology 130: 2786, 1983.
Institute for Clinical Immunology, Inselspital, Bern, Switzerland, and !Department of Clinical Immunology, Medical School Hannover, Hannover, FRG
381. Characterization of different epitopes on the T8 glycoprotein W. J. PICHLER, G. WOLFF-VORBECKt,
c. H. WALKER, and H. H. PETER!
To analyze the functional role of the T8 antigen, which is present on ca. 30 % of circulating T cells, ten monoclonal antibodies directed versus the T8 glycoprotein were studied for their inhibitory activity on MLR stimulated cytotoxic T cells and with respect to enzyme sensitivity of the epitopes recognized. The ten monoclonal antibodies react with different epitopes of the same antigen as shown by identical MW of ca. 76 KD, cocapping, and blocking of antibody binding. The epitopes recognized by the antibodies differed as they differ in their sensitivity to trypsin or papain and also because two epitopes were not expressed on rhesus macacae T lymphocytes. The inhibitory activity of the monoclonal antibodies was different, as those antibodies recognizing a highly trypsin sensitive region were almost always inhibitory, while the antibodies reacting with a relatively trypsin resistant region of the T8 glycoprotein inhibited the cytotoxicity in certain effector/target cell combinations only. Thus, the combined use of enzymatic digestion, phylogenetic comparisons and blocking studies may allow to define functionally distinct regions on the T8 glycoprotein and thus help to understand the function of the T8 'glycoprotein.
Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, P.O. Box 6120, Wiirzburg, FRG
382. Effect of monoclonal antibodies against rat leucocyte differentiation antigens on activation of in vitro selected encephalitogenic T line cells H.
J. SCHLUESENER and H. WEKERLE
Lymphocyte differentiation antigens can be more than mere descriptive markers of different lymphocyte subsets. Some of them are structures with definite functions corresponding to the differentiated status of the cell. Here we describe the effect of monoclonal antibodies against
16th International Leucocyte Culture Conference, Cambridge· 251 rat lymphocyte differentiation antigens and against rat MHC products on in vitro activation of encephalitogenic T cells. A Myelin Basic Protein (MBP) specific T cell line, vLe, was selected from naive, unprimed Lewis rat lymph node cells. The line cells display the rat T cell markers W3/13 and W3125 but are negative for OX 8 and OX 22. Upon activation with MBP presented by syngeneic accessory cells, the blasts transiently express la antigens as assessed by staining with monoclonal antibodies OX 3 and OX 6, which recognise polymorphic and monomorphic determinants of rat Ia respectively. la expression of the blast is concomitant with the ability to efficiently transfer lethal Experimental Allergic Encephalomyelitis (EAE) to normal adult syngeneic recipients (LDso: 1 x 106 cells/rat). The activation process can be blocked in vitro by addition of various monoclonal antibodies reactive with the responding T line cells or with the antigen presenting cells. The monoclonal antibodies OX 3 and OX 6 block T cell line activation at ascites dilutions of 1 :50,000, while antibody OX 17, reactive with a monomorphic determinant on the rat 1-E equivalent, is ineffective. This finding could suggest that responses against MBP in the Lewis rat are restricted solely by I-A region like gene products of the rat RT1.B complex. Furthermore, activation of T lymphoblasts could be blocked by antibodies directed against T cell differentiation antigens such as W3125, a putative analog of the human T4 marker.
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
383. The induction of non specific cytotoxic activity in human T-cell clones by antibodies against T3 H. SPITS, H. YSSEL, and E. DE VRIES The 13 antigen is a complex of at least 5 different proteins which is expressed on mature T cells. This complex appears to be involved in T-cell function. The activation of T cells by antigen can be mimicked by monoclonal anti-T3 antibodies. This is understandable since recent findings indicate that the receptor for alloantigen is associated with T3. Here we report that anti-T3 reagents, in addition to their capacity to induce proliferation and to block antigen specific cytotoxic activity mediated by cytotoxic T-cell (CTL) clones, induce non specific cytotoxic activity in cytotoxic T-cell clones. Furthermore we found that anti-T3 reagents were able to trigger cytolytic activity in T-cell clones, which were characterized as non cytotoxic antigen specific proliferating T cells. These results indicate that non cytotoxic T helper cells may also have a lytic machinery which can be triggered by anti-T3 reagents.
Laboratoire d'lmmunogenetique Humaine, C.H.U. Pitie-Salpetriere, 91, boulevard de I'Hopital, 75013 Paris, France
384. Biochemical evidence for clonotypic differences in antigen specific T cell clones T. TRIEBEL, P. DEBRE, and D.]. CHARRON Biochemical analysis of antigen specific T cell clones from a single individual may reveal clonotypic differences which could help to define new surface determinants such as differentia-
252 . 16th International Leucocyte Culture Conference, Cambridge tion antigens or antigen receptors. We obtained varidase (Var) or diphtheria toxoid (DT) specific cloned T cells from a single individual DR 6/7. These cloned cells were homogeneous in terms of membrane markers (OKT3+, OKT4+, OKTS-, RFB-l+, DR+) at a given stage of activation (3 days after stimulation with IL2). NP 40 totallysates of 35S methionine labelled cloned T cells were analysed by 2D-PAGE (IEF was used in the first dimension and a 10% SDS-PAGE in the second dimension). Three different spots were considered as present or missing in some of the clones tested after repeated electrophoresis of the same cell lysate. Spot 1 is very acidic with a MW of approximately 150 Kd. Spot 2 is slightly less acidic with a MW of 120 Kd. Spot 3 is very basic with a MW of 36 Kd. This biochemical heterogeneity demonstrate clonotypic differences in antigen specific T cell clones. The experimental approach described here may help to define important cellular proteins which could possibly reflect a given cellular function, specificity or differentiation stage.
Rotterdam Radio-Therapeutic Institute, The Netherlands
385. Human cytotoxic T cell clones show differential susceptability for
enhancement and/or inhibition of nonspecific cytolysis by lectins and anti-T3-MCA
R.
J. VAN DE GRIEND, C.
P. M. RONTELTAP, and R. L. H. BOLHUIS
The role of cell surface determinants as for instance recognized by the monoclonal antibodies (MCA) T3, T4, T8 etc. on the cytolytic activity of cytotoxic lymphocytes appears to be a complex one. In some T3+ T8+ clones we found that the HLA specific cytolytic activity can be blocked by OKT8 and by OKT3 (partially) MCAs, but that the cytolytic activity of other T3+ T8+ clones is not reduced at all. Two clones, Dll and G9, did not show cytolytic activity against Daudi cells but showed cytolytic activity against K562. However, when incubated with high concentrations of MCA against the T3 receptor, but not the T8 receptor, non-specific cytolysis against Daudi cells can be induced and the lytic activity against K562 augmented. Similar effects were obtained by preincubation of the effector cells with PHA or leucoagglutinin. In contrast, no cytolytic activity against Daudi cells was induced MCA in an T3+T4+ (n+20) allospecific cytotoxic clone, by OKT3 or OKT4 whereas those antibodies (partially) inhibited the cytolytic activity of this clone against the original stimulator cell. PHA had only a marginal effect on the cytolytic activity of this clone. Furthermore the cytolytic activity of OKT3-T4-T8-Tl1+ activated killer clones (recently established by us) is not augmented or inhibited by OKT3 or by lectins. The effects of lectin and OKT3 on the cytolytic activity of T-cell clones does apparently not depend on the presence or absence on the T3 marker but can also be different for T3+ clones varying from no effect on OKT3clones, an inhibitory effect on the anti class II T3+ T4+ clone (n+20), enhancement and/or induction of lytic activity on T3+ T8+ clone Dll or both inhibition and enhancement on T3+ T8+ clone G9. The cytotoxicity promoting activity of OKT3 on these clones thus resembles the effect of lectins, although quantitative differences exist. When compared with lectin, OKT3 has a stronger effect on the cytolytic activity against Daudi cells whereas the lysis of K562 is more augmented by PHA.
16th International Leucocyte Culture Conference, Cambridge . 253 Laboratory of Immunology, NIAID, NIH, Bethesda, Md 20205, U.S.A.
386. Inhibition ofT cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells P. WASSMER, C. CHAN, L. LOGDBERG, and E. M. SHEVACH
Two monoclonal antibodies (Mab) to distinct epitopes of the UT4 antigen (Mab, GK1.5 and 2B6) were tested for their capacity to block IL-2 secretion by concanavalin A (Con A) and antigen activated T-cell hybridomas. The T-cell hybridomas were MHC class II-restricted, insulin or ovalbumin specific, and of BALB/c or C57BL/6 origin. The induction of IL-2 production by antigen and syngeneic accessory cells (AC) was markedly susceptible to inhibition by Mab GK1.5. In general, 2B6 produced less inhibition of antigen-induced T cell activation. High concentrations of antigen could partially overcome the antibody induced inhibition. This result suggests a possible relationship between the susceptibility to blocking and the strength of the activation signal. We next examined the effect of anti-UT4 Mab on the ConA induced activation of the same panel of T cell hybridomas in an AC free culture. We found that both Mab were capable of producing marked inhibition of ConA induced IL-2 secretion although the susceptibility of the different hybridomas to blocking was more variable than that seen in the response to antigen. Again, an excess of the activation signal (higher concentrations of ConAl could partially overcome the inhibitory effect. In contrast to antigen driven IL-2 production, 2B6 was more potent than GK1.5 in blocking ConA induced activation. Because T-cell hybridomas were found to be Ia antigen negative as d_etermined by FACS analysis, the blocking of ConA activation could be demonstrated in a presumably MHC class II antigen free assay system. This observation is not consistent with the hypothesis that anti-UT4 antibodies inhibit T cell activation by interfering with interactions between UT4 and MHC class II products. An alternative explanation which is suggested by our findings is that the UT4 antigen interacts with the T cell receptor and plays a role in transmitting the activation signal as part of a multi-molecular complex. We cannot, however, exclude that anti-UT4 antibodies may inhibit T cell activation by more than one mechanism.