Lysis of hybridoma cells bearing anti-clonotypic surface immunoglobulin by clonotype-expressing alloreactive cytotoxic T cells.

A B cell hybridoma (Désiré-1) was derived which secreted and expressed at its cell surface immunoglobulin (Ig) specific for the antigen-specific T cell receptor (Ti) of an H-2Kb-specific alloreactive cytotoxic T lymphocyte (CTL) clone (KB5-C20). It was found that the CTL clone could lyse hybridoma Désiré-1, whereas it could not lyse hybridoma which expressed surface Ig (sIg) binding to other cell surface structures of clone KB5-C20 such as the H-2Kk molecule. Blocking of CTL-target cell interactions using monoclonal antibodies (mAb) indicated that the CTL-target cell interaction was inhibited with appropriate anti-H-2 mAb and by anti-Lyt-2 mAb when CTL-H-2Kb interaction was involved but not when CTL-sIg interaction was involved. The two types of interactions were inhibited by anti-LFA-1 mAb. The involvement of the CTL-Ti structure was necessary to obtain a lytic interaction between CTL and target cells, but a major histocompatibility complex product on the target cells did not need to be involved. Comparison of CTL-target cell inhibition with cold target cells or with anti-clonotypic mAb indicated that the Ti-sIg cellular interaction was of much higher apparent affinity than the Ti-H-2Kb cellular interaction. These results further suggest potential regulatory effects of CTL-B cell cross-idiotypic interactions.

Ontogeny of anti-self-trinitrophenyl cytotoxic T-cell responses.

The use of specific T helper cells (TH) has allowed the detection of anti-hapten plus self cytotoxic T-lymphocyte (CTL) precursors in cell populations unable to generate autonomous reactions (and considered as 'immature'). This led us to reevaluate the ontogeny of CTL precursors in different tissues in the presence of adult TH. It was found that thymocytes from newborn mice contained levels of CTL precursors comparable to those of adults and were dependent on TH throughout the donor's life. In the spleens, however, 3 periods could be distinguished: (a) from birth to 4 days after birth, no CTL precursors could be detected even in the presence of TH; (b) from the 5th day to the 2nd week after birth, CTL responses could be obtained only in the presence of TH; (c) after the 2nd week, autonomous responses could be obtained. No evidence for a suppressive mechanism was found in the spleen of the newborn mice, nor could lack of responsiveness be attributed to newborn accessory-cell malfunction. The data indicate that a limiting cell in the development of anti-hapten plus self CTL is the TH from the 4th to the 16th day after birth. The absence of CTL precursors in the spleen, but not in the thymus of neonates would indicate differential T-cell maturation in the two organs or late migration to the spleen.

Each of the two productive T cell receptor alpha-gene rearrangements found in both the A10 and BM 3.3 T cell clones give rise to an alpha chain which can contribute to the constitution of a surface-expressed alpha beta dimer.

The H-2 Kb specific cytotoxic T cell clone BM3.3 carries two productive TCR alpha gene rearrangements but expresses a single detectable TCR alpha beta chain combination on its surface. However, when separately analyzed by gene transfer, both productive alpha gene rearrangements are translated into alpha chains capable of pairing with the BM3.3 beta chain and of cell surface expression. Similar observations have been made with the products of the two productive alpha gene rearrangements previously identified in the A10 T cell clone. These observations contrast with those made for the two TCR alpha chains expressed in the MS202 T cell clone. In the latter instance, when analyzed by gene transfer, one of the alpha chains was unable to make a pair with the beta chain. Consequently, when considered with respect to the mechanisms of TCR allelic exclusion, our data suggest that the mere expression of a TCR alpha beta dimer on the surface of immature CD4+ CD8+ thymocytes may be necessary but not sufficient to shutdown further alpha gene rearrangements. Rather, the ability of a TCR alpha beta dimer to be positively selected may be needed to trigger the maturation of thymocytes to a stage devoid of recombinase activity. Alternatively, if non-dividing CD4+ CD8+ CD3+ small thymocytes do not experience secondary alpha-gene rearrangement, our data suggest that TCR alpha gene rearrangements are attempted quasi-simultaneously on both alpha alleles.

Interactions between the Thy-1 and T-cell antigen receptor pathways in the activation of cytotoxic T cells: evidence from synergistic effects, loss variants, and anti-CD8 antibody-mediated inhibition.

The relationship between activation of cytolytic T-lymphocyte (CTL) clones via the T-cell receptor (Ti) or the Thy-1 molecule was investigated. Anti-Ti and anti-Thy-1 monoclonal antibodies (mAb) can activate CTL clones to secrete interferon-gamma (IFN-gamma). Suboptimal doses of anti-Ti and anti-Thy-1 mAb, as well as suboptimal doses of two different anti-Thy-1 mAb, can synergize to activate T-cell clones. The addition of phorbol myristic acetate (PMA), which is not stimulatory by itself, can enhance the synergistic effect of mAb on IFN-gamma production. Although the Ti and Thy-1 molecules were not found associated at the cell surface, the results presented here indicate that these molecules are functionally associated. Use of Ti loss variants of a CTL clone confirms that Thy-1-mediated signaling is not an alternative to, but is dependent on the Ti-mediated activation pathway. Additionally, use of anti-Lyt-2/3 mAb, previously described as interfering with class I MHC-Ti binding and/or activation and, in some cases, with anti-Ti-mediated activation revealed that anti-Thy-1 mAb-mediated activation was also greatly reduced by the presence of Lyt-2/3-specific mAb. Thus the interaction between Thy-1 and Ti might also involve Lyt-2 (Lyt-3) molecules.

T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A.

Fas/APO-1 is a member of the tumor necrosis factor receptor family of proteins that induces apoptosis when cross-linked with monoclonal antibody (mAb) or with its physiological ligand. Recently, both a perforin-based and a Fas-based mechanism have been proposed to account for T cell-mediated cytotoxicity. In the present study we used a murine CD8+ cytotoxic T lymphocyte (CTL) clone (KB5 C20) specific for H-2Kb and a T cell receptor (TcR)-negative variant of the same clone (2005-D4) to test (i) whether the same cell can exert both cytotoxic effector mechanisms and (ii) the role of TcR engagement in the induction of Fas-based cytotoxicity. We demonstrate that both the TcR+ and TcR- clones were able to express the Fas ligand after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and that TcR engagement of the KB5.C20 clone by means of antigen-bearing cells or of its anticlonotypic mAb (Désiré-1), which leads to Ca(2+)-dependent, presumably perforin-based, cytotoxicity, was also able to induce Fas-based cytotoxicity. In addition, using inhibitors we investigated the signal transduction pathway(s) involved in the induction of Fas-based cytotoxicity and expression of the Fas ligand mRNA in the CTL clones. The involvement of src-like protein tyrosine kinases (PTK) in Fas ligand induction through TcR engagement, was strongly suggested by inhibition with the src-like PTK inhibitor herbimycin A. Inhibition of Fas ligand induction by genistein, a more general TPK inhibitor, even upon stimulation by PMA plus ionomycin, suggested the possible involvement of PTK activities downstream of protein kinase C (PKC) in Fas ligand induction in CTL. Finally, the implication of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in Fas ligand induction was demonstrated by the partial inhibition of Fas ligand induction with cyclosporin A. Thus, in CTL clones, Fas ligand expression is inducible by TcR engagement through a pathway similar to that involved in expression of some lymphokine genes.

Developmental control of T-cell receptor internalization.

Since TCR/CD3 modulation may be involved in induction of T cell tolerance to self antigens, we compared ligand-induced TCR/CD3 internalization by a CTL clone and by immature thymocytes and mature T cells from mice bearing the same TCR alpha beta as transgene. The ligand used is a monoclonal antibody (mAb) specific for the receptor expressed by the clone and transgenic mice (anti-Ti mAb). CD8+ splenocytes triggered by anti-Ti mAb internalize the ligand-TCR/CD3 complex at a low rate, through a mechanism inhibited by the protein tyrosine kinase (PTK) inhibitor genistein and by staurosporine, a potent but non selective protein kinase C (PKC) inhibitor. This pattern of inhibition was similar to that observed in the CTL clone. Anti-Ti mAb induced TCR/CD3 internalization in CD4+CD8+ thymocytes at a high rate, through a mechanism which was insensitive to either genistein or staurosporine. In the CTL clone, genistein was shown to inhibit TCR/CD3 surface redistribution preceeding internalization. To characterize the PTK possibly involved in this step, we analyzed TCR/CD3 associated kinases in mature T splenocytes and thymocytes. Kinase activities present in anti-Ti mAb immunoprecipitates phosphorylated the CD3 components gamma, delta, epsilon, and zeta in both cell types although the intensity was stronger in splenic than in thymocyte extracts, whereas the phosphorylation of 70, 14 and 12kD substrates was more pronounced in thymocytes than in splenocytes. Comparable amounts of CD3 components were coprecipitated with and phosphorylated by p56lck and p59fyn respectively, in both cell types.

Monoclonal antibodies against an H-2Kb-specific cytotoxic T cell clone detect several clone-specific molecules.

Monoclonal antibodies (mAb) were prepared from the spleen of a BALB.K mouse immunized with a H-2Kb-specific alloreactive cytolytic T cell (CTL) clone of B10.BR origin, KB5-C20. Six of these mAb were selected for specific binding to the immunizing clone. Only one of these mAb, Désiré-1, inhibited the CTL activity of clone KB5-C20. Functional and biochemical evidence showed mAb Désiré-1 to be anti-clonotypic (anti-Ti). This mAb immunoprecipitated from lysates of clone KB5-C20 a protein that migrated at 80 KD in nonreducing conditions and at 40 to 45 KD in reducing conditions. Analysis in NEPHGE resolved the latter into an acidic chain of 43 KD and a basic chain of 40 KD. An additional protein was coprecipitated at 25 KD in nonreducing and 27 KD in reducing conditions, which was distinct from the Thy-1 antigen. Expression of the clonotype defined by Désiré-1 was found on clone KB5-C20 and on clone KB5-A7 originating from the same B10.BR T cell population. These two clones exhibited a similar pattern of reactivity on H-2Kbm mutant target cells. Another clone (KB5-C1) from the same B10.BR T population showed a distinct reactivity pattern on the H-2Kbm mutant target cells and did not express the Désiré-1 clonotype, nor did Con A-activated B10.BR blast cells or H-2Kb-specific clone BM3.3 of CBA/J origin. Among the five other selected mAb, one reacted with the three Kb-specific CTL clones of B10.BR, but not with the Kb-specific clone of CBA origin, whereas the last four mAb bound only to clone KB5-C20. None of these mAb could immunoprecipitate the Ti alpha/beta chains. Ti modulation induced by mAb Désiré-1 on clone KB5-C20 did not induce comodulation of the structures bound by the other five mAb on clone KB5-C20, thus indicating that these structures were not part of the Ti-T3 complex. These results characterize a clonotypic mAb reacting with an H-2Kb-specific CTL clone that will be useful in future studies. They also indicate that screening for clone-specific mAb by selective binding assays may select for mAb against as yet undefined structures, some of which exhibit an apparently clone-specific distribution.

Phosphorylation of T-cell antigen receptor-associated proteins: correlation with activation for killing and/or for gamma-interferon production by a cytolytic T-cell clone.

The activation-induced phosphorylation of T-cell antigen receptor (Ti)-associated proteins was investigated in order to analyse possible signal-transduction mechanisms leading to two distinct effector functions of a mouse cytolytic T-cell clone (KB5.C20): target cell killing (independent of protein synthesis) and de novo production of gamma interferon (gIFN; dependent on gIFN gene expression). Ti-associated T3-like proteins were first identified by immunoprecipitation of 125I-labelled cell surface proteins from 1% digitonin lysates of clone KB5.C20 by 1- and 2-dimensional (non-reduced (NR)/reduced (R)) gel electrophoresis. In addition to the alpha and beta chains of the Ti (NR: 80-Kd; R: 43 and 40 Kd), two doublets of 35-37 Kd (NR) and 32-34 Kd (NR) leading to bands of 25, 16 and 14 Kd (R) were identified, as well as three bands (25, 23 and 22 Kd (NR)) leading to 27-, 25- and 21-Kd bands (R). Activation of clone KB5.C20 (prelabelled with 32P-orthophosphate) with either anti-Ti mAb or exposure to both ionomycin and phorbol myristic acetate (PMA) induced the phosphorylation of 21- and 25-27-Kd (R) Ti-associated proteins, whereas exposure to either ionomycin or PMA alone induced only weak phosphorylation of 21-Kd (R) components. A weak phosphorylation of 32- and 34-Kd Ti-associated proteins was sometimes observed after stimulation with anti-Ti mAb. Functional studies suggested that activation for gIFN production was observed only when both the 21- and 25-27-Kd proteins were phosphorylated, whereas activation for killing (when measured by PMA-induced non-specific killing) could occur in conditions where no phosphorylation of the 25-27-Kd protein was detected.

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli.

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanically-induced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.

Role of CD3gamma and CD3delta cytoplasmic domains in cytolytic T lymphocyte functions and TCR/CD3 down-modulation.

TCR engagement leads to down-modulation of TCR/CD3 complexes from the T cell surface. The importance of this effect in T cell physiology is unknown. Here, we characterized a CTL clone deficient in TCR/CD3 surface expression that had lost both CD3delta and CD3gamma mRNA, allowing us to address the role of these chains in the assembly, signaling, and dynamics of the TCR/CD3 complex. Expression of either CD3delta or CD3gamma alone failed to reconstitute surface expression of the TCR/CD3 complex, but reconstitution with a cytoplasmically truncated CD3delta (delta t) and a native (gamma) or cytoplasmically truncated (gamma t) human CD3gamma led to reexpression of TCR/CD3 complexes in both cases. This indicated that CD3delta and CD3gamma assume specific functions in TCR/CD3 assembly independently of their cytoplasmic domains. The delta t gamma t variant specifically killed target cells, expressed the IFN-gamma gene in response to Ag, and produced TNF-alpha in response to anti-CD3 mAb, but it was affected in CD3 ligand-induced TCR/CD3 down-modulation. Both PMA- and CD3 ligand-induced TCR/CD3 down-modulation were defective in the delta t gamma t variant, whereas the delta t gamma variants were unaffected, and previously described delta gamma t variants were affected only in PMA-induced down-modulation. Specific protein kinase C (PKC) inhibitors indicated that PMA- but not CD3 ligand-induced down-modulation was dependent on PKC activity. Thus, amino acid sequences present in either the CD3delta or CD3gamma cytoplasmic domain control ligand-induced TCR/CD3 down-modulation, and neither these sequences nor this property are required for cytolysis and IFN-gamma gene expression in response to Ag.

Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution.

Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.

TCR-associated zeta-Fc epsilon RI gamma heterodimers on CD4-CD8- NK1.1+ T cells selected by specific class I MHC antigen.

The origin of autoreactive CD4-CD8- T cells is largely unknown. In TCR transgenic (Tg) mice expressing the cognate class I MHC antigen, CD4-CD8- T cells differed depending on characteristics of Tg-TCR/antigen interaction. Tg-TCR/CD3lo CD4-CD8- T cells expressing the NK1.1 marker were observed only for a Tg-TCR whose stimulation by antigen was independent of CD8. Unlike normal T cells, which have essentially TCR-associated zeta homodimers, these cells had a high proportion of TCR-associated zeta-Fc epsilon RI gamma heterodimers. They were also characterized by an unusually high content of Fc epsilon RI gamma mRNA and low content of mRNA encoding CD3 epsilon, CD3 gamma, CD3 delta, and zeta. Based on their phenotype and selection requirements, it is proposed that CD4-CD8- thymic precursor cells can be driven along the CD4-CD8-NK1.1+ pathway following coreceptor-independent TCR signaling at an intrathymic stage when Fc epsilon RI gamma and CD3 components are coexpressed.

Antigen-induced TCR-CD3 down-modulation does not require CD3delta or CD3gamma cytoplasmic domains, necessary in response to anti-CD3 antibody.

We studied cytotoxic T lymphocyte (CTL) clones expressing cytoplasmic domain-deleted CD3delta and CD3gamma chains. These cells retained efficient antigen-specific cytolysis. Because the cytoplasmic domains of native CD3delta and CD3gamma chains contain a dileucine-based and a tyrosine-based motif thought to be important for receptor endocytosis, we compared TCR-CD3 down-modulation on the CTL clones expressing or not these domains. We found that antigen-induced TCR-CD3 down-modulation was not dependent on either the CD3delta or CD3gamma cytoplasmic domains. This contrasts with phorbol ester- and anti-CD3 mAb (soluble or plastic-coated)-induced TCR-CD3 down-modulation, that are respectively dependent on CD3gamma and on either CD3delta or CD3gamma cytoplasmic domains, suggesting that differences may exist between the mechanisms of TCR-CD3 down-modulation in response to the three stimuli. TCR-CD3 down-modulation in response to antigen was demonstrated by confocal microscopy to be associated with TCRbeta chain internalization, whether CD3delta and CD3gamma were native or truncated. Inhibition by the protein tyrosine kinase inhibitor PP1 of TCR-CD3 down-modulation in response to antigen was also similar whether CD3delta and CD3gamma cytoplasmic domains were present or not. These properties of receptor down-modulation are discussed with respect to the requirements for TCR engagement on antigen-presenting cells.

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity.

In preparation for functional analyses, a study of the binding of H-2Kb-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2Kbm mutant cells and on FOR-treated H-2Kb cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2Kb-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2b cells as indicated by cold target inhibition studies. The H-2Kb-specific CTL were probably reactive with "conformational" determinants of H-2Kb, which are dependent on the integrity of both the alpha 1 and the alpha 2 domains of the H-2Kb molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the alpha 1 domain without affecting conformational-type CTL determinants.

Antigenic and genetic parameters in the stimulation and in the lytic phases of anti-hapten + self cytotoxic T cells and their derived clones: role of the T helper cell.

Requirements for stimulation of cytotoxic T cells (CTL) and for their lytic recognition have been compared in T cell lines repeatedly stimulated with trinitrobenzene sulfonate-treated syngeneic murine spleen cells. Differences were observed between the requirements for cells to stimulate or to be lysed by the CTL, which included: (a) the expression of major histocompatibility complex (MHC = H-2) encoded allelic products, and (b) the hapten density. Propagation of the CTL within the line required I-A intra-H-2 homology between hapten-treated stimulation cells and the line cells, whereas the lytic interaction required H-2K region homology between hapten-treated target cells and CTL. The hapten density requirement was analyzed for a responder (H-2k) and a non-responder (H-2b) strain to low hapten density modified syngeneic cells. This property was found to be a characteristic of the lytic phase rather than of the stimulation of CTL. CTL clones could be derived by growing the line cells under conditions of limiting dilution in the presence ot T cell growth factors. Such CTL clones were unable to be stimulated by their target antigens and were dependent on T cell growth factors for their propagation. These results are discussed in terms of the dependence of the development and growth of CTL on T helper cells.