We studied cytotoxic T lymphocyte (CTL) clones expressing cytoplasmic domain-deleted CD3 delta and CD3 gamma chains. These cells retained efficient antigen-specific cytolysis, Because the cytoplasmic domains of native CD3 delta and CD3 gamma 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 downmodulation was not dependent on either the CD3 delta or CD3 gamma cytoplasmic domains. This contrasts with phorbol ester- and anti-CD3 mAb (soluble or plastic-coated)-induced TCR-CD3 downmodulation, that are respectively dependent on CD3 gamma and on either CD3 delta or CD3 gamma cytoplasmic domains, suggesting that differences may exist between the mechanisms of TCR-CD3 downmodulation in response to the three stimuli. TCR-CD3 down-modulation in response to antigen was demonstrated by confocal microscopy to be associated with TCR beta chain internalization, whether CD3 delta and CD3 gamma 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 CD3 delta and CD3 gamma 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.

The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g(7) congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.

The use of flow cytometry to detect apoptotic thymocytes is now well established. We have further developed the technique of Hoechst 33342 vital staining to identify discrete stages of murine thymocyte apoptosis (induced by 37 degrees C culture), in conjunction with propidium iodide (PI), cell scatter profile, and surface marker analysis. The first detectable stage was an increase in Hoechst fluorescence without any change in plasma membrane permeability (measured by PI staining). At this early stage thymocytes had already reduced in size, fragmented their DNA, and for the predominant CD4+ CD8+ double positive population, reduced expression of CD4 and CD8. Subsequent to this stage thymocytes continued to reduce in size and decrease expression of CD4 and CD8, though this was accompanied by an increase in membrane permeability. This technique was applied to an in vitro antigen-specific deletion system, where apoptosis of T cell-receptor-transgenic thymocytes was induced upon presentation of self-antigen. Although self-antigen-induced apoptotic thymocytes showed similar characteristics to those undergoing spontaneous apoptosis, there was a significant population of nonapoptotic CD4+ 8+ thymocytes that also had reduced expression of CD4 and CD8. Therefore, we have been able to show that the reduced expression of CD4 and CD8 is not limited to apoptotic thymocytes.

The TCR is composed of two chains (alpha/beta) containing variable regions associated at the cell surface with invariant chains (CD3 gamma-, delta-, epsilon-, and zeta/eta-chains). The latter control assembly and surface expression of the TCR/CD3 complex, as well as its cytoplasmic association with signal transduction relays. In differentiated CTL, stimulation through the TCR leads to the transcriptional activation of genes coding secreted cytokines such as gamma-IFN as well as transcription-independent activation of the lytic machinery. It is not known which of the CD3 components is necessary to transduce the required signals. CD3 gamma- and delta-chains have high sequence homology, in particular in their cytoplasmic domain, and it has been proposed that alpha-beta-gamma-epsilon-zeta and alpha-beta-delta-epsilon-zeta may be expressed and function in signal transduction independently. Here, we characterize a CTL clone that has selectively lost expression of the CD3-delta mRNA. This results in expression of partial CD3 complexes devoid of TCR-alpha-beta chains at the surface of the clone, which are not functional for activation of cytolysis or for gamma-IFN production. Transfection of the clone with either the native or a cytoplasmic exon-deleted CD3-delta gene restores full TCR/CD3 surface expression as well as Ag- or CD3-mediated activation for killing and for gamma-IFN production, indicating that the CD3-delta chain is essential for surface expression of the TCR-alpha-beta, but that the CD3-delta cytoplasmic portion is not required either for complex assembly or for signal transduction involved in the functions studied.

The extent of peripheral clonal deletion of the T cell antigen receptor (TCR) from a CD8-dependent cytotoxic T lymphocyte of H-2k origin with alloreactivity for H-2Kb was measured in a TCR transgenic (Tg) model by use of a clonotype-specific mAb (anti-Ti mAb). Deletion varied depending on whether the TCR Tg was expressed in mice heterozygous (H-2k x b) or homozygous (H-2b x b) for the H-2Kb antigen. CD8 surface staining and functional analyses of peripheral T cells stimulated with polyclonal activators in bulk culture or by limiting dilution confirmed that in the H-2b x b mice few Ti+ cells were generated as measured by anti-Ti mAb-dependent target cell killing; while such cells could readily be detected in the H-2k x b mice. The latter maintained non-responsiveness to H-2Kb, as measured by cytolysis of H-2Kb-expressing tumor target cells, due to their down-regulation of surface CD8 expression. The question of whether the difference between H-2b x b and H-2k x b mice was due to the influence of positive selection imposed by the k haplotype in the H-2k x b hybrid, or to the lower antigen density in heterozygous than homozygous mice was addressed by analysis of H-2b x d Tg mice, H-2d being a non-selecting haplotype. Results obtained were similar for H-2b x d and H-2k x b Tg mice, suggesting that the density of the H-2Kb antigen may be one of the parameters controlling the extent of clonal deletion in the thymus.

Biology of the CellVolume 76, Issue 2 p. 250-250 Detection of normal and apoptotic thymocytes by hoechst 33342 with simultaneous three colour surface phenotype analysis Curnow John, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this authorBarad Marc, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this authorBrun-Roubereau Nicole, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this author Curnow John, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this authorBarad Marc, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this authorBrun-Roubereau Nicole, Centre d'immunologie de Marseille-Luminy, Case 906, 13288 Marseille, Cedex 9, FranceSearch for more papers by this author First published: 1992 https://doi.org/10.1016/0248-4900(92)90331-TAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume76, Issue21992Pages 250-250 RelatedInformation

The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti‐TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12‐myristate 13‐acetate (PMA). After binding of the anti‐TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti‐TcR mAb. PMA‐induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC‐depleted cells or in the presence of the PKC inhibitor staurosporine. Anti‐TcR mAb‐induced internalization was maintained in PKC‐depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti‐TcR mAb Fab fragment is non‐stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC‐depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA‐induced phosphorylation of γ and ζ subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross‐linking of the TcR via plastic‐coated anti‐TcR mAb led to phosphorylation of CD3 γ and ϵ and also of ζ, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti‐TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.

To analyze the expression of the interleukin (IL)2 gene at the single cell level, we have constructed a chimeric gene in which the regulatory sequences from the mouse IL 2 gene were fused 5' proximal to the coding region of the Escherichia coli β‐galactosidase (lacZ) gene. Once stably introduced into a T cell hybridoma, the IL 2‐lacZ reporter gene was shown to display a pattern of induction similar to the one observed for the resident IL 2 gene. Two points emerged from monitoring IL 2 expression at the single cell level. First, upon activation the expression of the IL 2‐lacZ gene appears asynchronous. Second, at the peak of induction the percentage of β‐galactosidase+ cells never reached 100% and the level of β‐galactosidase reached among positive cells was highly heterogeneous within a cloned population of T cells. In combination with the possibility to sort viable lymphocytes according to lacZ expression, the IL 2‐lacZ reporter gene described herein should provide a way to isolate somatic cell mutants deficient in signal transduction by the T cell antigen receptor complex.

The regulation by IFN of the expression of HLA-B7 and HLA-A3 class I molecules was studied in Jurkat human T lymphoma cells, HHK EBV-transformed human B lymphocytes, and murine HLA-B7 HLA-A3 co-transfected L fibroblasts. Jurkat cells express constitutively low level of HLA class I molecules and treatment with human IFN resulted in preferential increase of the expression of HLA-B7 molecules, the expression of the HLA-A3 molecules being relatively unchanged. Similar treatment of HHK cells, which express constitutively large amount of HLA class I molecules, resulted in a marginal increase of the expression of both HLA-B7 and HLA-A3 molecules. HLA-B7 HLA-A3 co-transfected L cells express relatively low level of HLA class I molecules, expression of both however was significantly increased after treatment with murine INF-alpha, the augmentation being more accentuated for HLA-B7 molecules. In all cases, variations of cell surface expression were related to parallel modifications of the level of HLA-B7 and HLA-A3 RNA transcripts. Important nucleotide differences exist between the IFN consensus sequences associated with the HLA-B7 and HLA-A3 class I genes. Using oligonucleotides corresponding to these sequences two patterns of retarded bands were observed by the gel mobility shift assay, suggesting that the IFN-mediated differential regulation of the expression of the HLA-B7 and HLA-A3 genes could be due to different nuclear regulatory factors.

The cytolytic responses of DBA/2 mice against syngeneic transfected P815 mastocytoma cells expressing either membrane-associated (HLA-Cw3) or -secreted hybrid (HLA-Cw3 x H-2 Q10b) molecules were compared. In spite of the absence of serologically detectable hybrid molecules on their plasma membrane, cells secreting these molecules elicited a CTL response similar to that of cells expressing the membrane associated HLA-Cw3 molecules, in terms of both MHC-restriction and peptide specificity. Together with the observation that syngeneic mice were capable of rejecting the injected secreting cells, these results imply that secreted HLA class I molecules can function as minor histocompatibility Ag and suggest that processing of both the membrane-bound and the -secreted forms of a protein may follow common or overlapping pathways.

Sequential transfections of P815 murine mastocytoma cells with class I gene encoding either HLA-Cw3, HLA-A3, or HLA-B7 H chain and subsequently with a human beta 2-microglobulin gene were performed to evaluate the relative efficiency of human and murine beta 2-microglobulins in promoting the cell-surface expression of HLA-class I molecules. A 6-, 11-, and 40-fold specific enhancement of the cell-surface expression of HLA-Cw3, HLA-A3, and HLA-B7 molecules, respectively, was observed in cells co-transfected with human beta 2-microglobulin gene. This effect was attributed to a more efficient association of HLA H chains with human than with murine beta 2-microglobulin, which apparently allowed a more rapid transport of the HLA molecules from the endoplasmic reticulum to the Golgi apparatus.

1. Rabbit retinas were isolated and subjected in vitro to shifts between light and darkness in the presence or absence of four concentrations of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Changes in the rate of cyclic GMP hydrolysis (determined by 18O labelling of guanine nucleotide alpha-phosphoryls) and in total cyclic GMP content (determined by radioimmunoassay) were compared with the changes in the electrical potential across the retina. The experiments were designed so that the changes in potential would reflect changes in the light-sensitive conductance of the photoreceptors. 2. IBMX at 27-730 microM caused dose-related reductions in cyclic GMP hydrolysis in both light and darkness. The reductions in hydrolysis were associated with almost equal reductions in synthesis, so that there was little increase in the total content of cyclic GMP despite large changes in its metabolic flux. 3. Shifting from light (2.3 x 10(3) photons microns-2 s-1) to darkness also caused large reductions in the metabolic flux of cyclic GMP, with little increase in its total content. 4. Reductions in cyclic GMP flux were always associated with increases in the vitreous-positive transretinal potential, which was used as a measure of photoreceptor outer segment conductance, and the inverse correlation between flux and potential was closely maintained (r = 0.98) under all conditions examined. The correlation between total cyclic GMP content and transretinal potential was much less close. 5. Since IBMX and darkness acted similarly and additively, the combination of IBMX and darkness caused large decreases, of up to 21-fold, in cyclic GMP flux and large increases, of up to 23-fold, in the transretinal potential. 6. Kinetic analysis of the data indicated that the great majority (about 95%) of the light-sensitive conductance was closed under physiological conditions in darkness. 7. The data appear to be consistent with a system in which much of the cyclic GMP is bound, in which the binding is increased by light, and in which the free cyclic GMP acts co-operatively with a Hill coefficient of 3 to open outer segment conductance and to inhibit guanylate cyclase.

This study demonstrates that cytolytic T-cell lines exhibit progressive in-vitro modifications of their phenotype and of their growth behaviour and may use different pathways for their multiplication. Comparing three established cell lines, we firstly demonstrated that the expression of LFA-I is stable but the Lyt 2, 3 is rapidly lost. In this case, a high lectin-dependent cytotoxicity appears. Secondly, we demonstrated that two of the cell lines used the interleukin 2-interleukin 2 receptors (IL-2-IL-2R) binding pathway. Two different monoclonal antibodies showed that the IL-2 receptors distribution does not correlate with the number of functional sites which determines the IL-2 requirement. In contrast, the third cell line, although bearing high levels of IL-2 receptors, grows without the addition of IL-2; this cell growth is not inhibited by anti-IL-2 receptors monoclonal antibodies. Thirdly, it appears that the new property of IL-2 independence is associated with acquisition of the simultaneous capacity to induce tumour grafts in nude mice. As it has been recently reported that cytolytic T-lymphocytes against tumour cells could be promising immunotherapeutic agents, the spontaneous malignant transformation of such CTL lines should be taken into account before using them for adoptive immunotherapeutic purposes.

Glycosyl-phosphatidylinositol (G-PI) has been shown to serve as membrane anchor for cell surface molecules such as Thy-1, Ly-6-controlled ThB and Qa antigens. Here, we present several lines of evidence indicating that the hematopoietic cell lineage (i.e. thymocytes, B cell subset and red blood cells) marker defined by the rat monoclonal antibody J11d is also a G-PI-linked structure. First, surface expression of the J11d-defined molecules, and that of the related antigen B2A2, was found to be specifically reduced by treatment of thymocytes and B lymphoma or hybridoma cells with excess of Staphylococcus aureus PI-specific phospholipase C; this enzyme also solubilizes a 35-40-kDa material from erythrocyte microsomal membranes corresponding to the predominant J11d-reactive red cell surface molecules. Second, Thy-1- mutants of the BW5147, T1M1, S1A or S49 murine T lymphoma cells of the complementary classes A, B, C and E (i.e. shown to be defective in the enzymatic machinery that posttranslationally modify Thy-1 molecules) also lack J11d, or express it at a very low level. Although directed at a G-PI-linked structure, the J11d monoclonal antibody, unlike other reagents to Thy-1 or Ly-6-controlled antigens, failed to induce thymocyte proliferation even in the presence of phorbol myristate acetate and cross-linker monoclonal antibody.