Receptors and signals in early thymic selection.
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Biomedical subjects
K Eichmann
Publications and source records attributed to K Eichmann.
Regulation of thymocyte development through CD3: functional dissociation between p56lck and CD3 sigma in early thymic selection.
We studied the extent of functional linkage between CD3 sigma and p56lck in pre-TCR-dependent thymocyte development. Differentiation of DN to DP cells was examined by treatment of RAG2/CD3 sigma and RAG1/p56lck double-deficient mice with anti-CD3 epsilon antibodies. The results suggest that CD3 sigma has no specific role in this maturation step, but may be important for amplification of signaling through the pre-TCR. In contrast, p56lck is the main protein tyrosine kinase associated with signaling through the pre-TCR-CD3 complex. In DP thymocytes, the Ca2+ response to anti-CD3 epsilon was totally abolished in CD3 sigma-I-but only reduced in p56lck-I-mice, and in vivo responses to anti-CD3 epsilon differed from one another. Thus, CD3 sigma and p56lck are functionally not tightly associated and their deficiencies cause distinct developmental defects.
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In vitro generation of lymphoid precursors from embryonic stem cells.
Murine embryonic stem (ES) cells represent a model system for studying certain aspects of hemopoiesis because they can differentiate in vitro into several cell types, including those of the hemopoietic system. We developed cell culture conditions in which ES cells undergo hemopoietic differentiation in a low-oxygen (5% O2) atmosphere without additional exogenous factors. After 15-20 days of culture under these conditions, cells bearing surface markers found on cells of the lymphoid lineage (Thy1+, Pgp-1+, c-kit+ and B-220+) were detected. After 13-15 days, transcripts for the recombinase activating genes (RAG) 1 and 2, interleukin (IL) 7, IL-7 receptor and c-kit were expressed. We also investigated rearrangements of the immunoglobulin (Ig) heavy and light chain and the T cell receptor (TCR) loci. After 15 days of differentiation, we detected DJH gene rearrangement with N-region diversity. Productive VHDJH rearrangements are found after 20 days, paralleled by V Kappa J Kappa recombinations indicating a developmental stage comparable, at least, with that of pre B cells. Rearrangements of TCR gamma as well as delta chain segments were also observed, but no TCR beta chain rearrangement. These results demonstrate that ES cells reproducibly generate lymphoid cells in vitro.
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Streptavidin-tricolor is a reliable marker for nonviable cells subjected to permeabilization or fixation.
Cell death is normally accompanied by loss of integrity of the cell membrane. Concomitant loss of osmotic pressure and permeability for DNA binding dyes like propidium iodide make it possible to distinguish viable from nonviable cells. However, after permeabilization for intracellular staining or after fixation of the cells, we find that propidium iodide leaks out of nonviable cells and is transferred to formerly viable cells. Cell size cannot be used for examining viability in permeabilized or heterogeneous cell populations. Here we show that streptavidin-tricolor enters specifically and irreversibly into dead cells, and is not transferred to formerly viable cells after fixation or permeabilization. Therefore, streptavidin-tricolor can be a useful dead-cell marker in experimental situations where conventional methods fail to distinguish between viable and nonviable cells.
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The mouse bone marrow stroma cell line S17 supports growth at limiting cell numbers of EBV-transformed human lymphoblastoid B cell lines.
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Human immune response to HIV-1-Nef. I. CD45RO- T lymphocytes of non-infected donors contain cytotoxic T lymphocyte precursors at high frequency.
The immune response of peripheral blood lymphocytes (PBL) of non-exposed human individuals to the Nef protein of HIV-1 was studied. Nef is a regulatory protein of HIV which is immediately expressed after infection and which seems to be important in the pathogenicity of HIV. Nef may therefore serve as a potential target for effective immunity against HIV infection. Epstein-Barr (EBV)-transformed lymphoblastoid B cell lines (LCL) were established from four healthy young seronegative adults and transfected with the Nef gene. These cells served as stimulator cells for autologous PBL in vitro and as target cells for CTL. CTL responses were readily generated against Nef-transfected LCL, consisting of Nef-specific and putative EBV-specific CTL. Nef-specific CTL were generated exclusively from CD8+ cells and were MHC class I restricted. Since a vigorous Nef-specific CTL response in non-infected individuals was unexpected, CTL precursor frequencies were determined by limiting dilution analyses in non-fractionated PBL and in PBL separated into the CD45RO- (naive) and CD45RO+ (memory) T cell populations. As expected, the putative EBV-specific CTL precursors were predominantly found in the CD45RO+ subset at frequencies typical for memory T cells. Nef-specific CTL precursors, in contrast, were found predominantly in the CD45RO- population, at even higher frequencies of approximately 1/1000-1/3000. Nef may thus display either an unusually high number of immunogenic peptides or a limited number of peptides presented in a very efficient way, so that many T cells including low affinity cells, would be triggered.
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Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon.
Thymic repertoire selection requires the expression of the alpha beta CD3 T-cell receptor (TCR) together with the coreceptors CD4 and CD8. The appearance of CD4 and CD8 on thymocytes is the hallmark of a complex maturation step, accompanied by downregulation of the interleukin 2 receptor (IL-2R) alpha chain, arrest of rearrangement (i.e., allelic exclusion) of the TCR beta-chain locus, a burst of cell divisions, and reduction in cell size. This maturation step is inhibited in TCR beta-chain-deficient mouse strains and may depend on surface expression of an immature TCR complex containing CD3 and TCR beta chains but no TCR alpha chain. Here we show that the CD4+8+ double-positive (DP) stage can be induced by treatment of fetal thymic organ cultures with anti-CD3 epsilon monoclonal antibodies in several TCR beta-chain-deficient mouse strains: severe combined immunodeficient (scid) mice, mice carrying a mutation in the recombination activating gene 1 (Rag-1), or mice carrying a deletion in the TCR beta-chain locus itself. These findings suggest that CD3 epsilon is expressed on the thymocyte surface independent of and prior to the TCR beta chain. The data are consistent with the notion that in wild-type mice the DP stage is induced by transmembrane signaling through an immature CD3-TCR beta-chain complex, which can be bypassed by crosslinking of CD3 epsilon alone.
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Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.
Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.
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Regulation of thymocyte development through CD3. I. Timepoint of ligation of CD3 epsilon determines clonal deletion or induction of developmental program.
Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)-2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR-beta chain homodimers and CD3. Here we show that early addition of anti-CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR-beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.
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Lineage relationships of the fetal thymocyte subset that expresses the beta chain of the interleukin-2 receptor.
The beta chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R beta+ thymocytes are gamma delta cells. The precursor-progeny relationships of fetal IL-2R beta+ thymocytes to the alpha beta T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2R beta is expressed on a subset of very immature cells, along with high amounts of Pgp1 and Fc gamma RII/III, partially preceding the expression of intracellular CD3 epsilon. IL-2-R beta disappears before expression of IL-2R alpha. IL-2R beta+ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR) alpha beta+, TCR gamma delta+, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2R alpha+ day 15 fetal thymocytes. These data suggest that IL-2R beta expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.
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Intracellular processing of hapten-modified protein for MHC class I presentation: cytoplasmic delivery by pH-sensitive liposomes.
Phagocytic or endocytic uptake of pH-sensitive liposomes has been shown to result in the release of entrapped material into the cytosol. This system can therefore be applied to the targeted delivery of protein antigens into the MHC class I presentation pathway of antigen-processing cells. We have used trinitrophenyl (TNP)-modified chicken ovalbumin encapsulated in liposomes to examine the intracellular processing of haptenated proteins and the presentation of TNP-modified peptides to MHC class I-restricted hapten-specific CTL. Here, we demonstrate for the first time that hapten-modified proteins can undergo intracellular processing by macrophages, that similar peptides are produced in the form of unmodified or haptenated derivatives, and that TNP-peptides are transported to the cell surface and presented to class I-restricted CTL via the ER/Golgi pathway. This system can now be used to study T-cell responses to naturally processed hapten-conjugated peptides in vitro and in vivo.
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Constitutive expression of transgenic heat stable antigen (mCD24) in lymphocytes can augment a secondary antibody response.
The murine heat stable antigen (HSA, mouse CD24) is a glycosyl phosphatidylinositol-anchored cell surface protein which is primarily expressed in immature but not mature cells of several hematopoietic lineages and in neuronal tissue. The function of HSA is not known but there is evidence in lymphocytes that it is involved in cell adhesion and in cell activation. We examined the effect of constitutive HSA expression on the maturation and on the function of B and T cells in transgenic mice. Transgenic HSA was strongly expressed throughout all stages of T cell maturation without changes in absolute cell number or proportions of subpopulations both in the thymus and in the periphery. The size of the B cell compartment was also unchanged. Thus, we conclude that in the T lineage the loss of HSA expression is not mandatory for maturation. On the functional level, two parameters of immune function were measured. When the ability to activate peripheral T cells expressing transgenic HSA was tested in a mixed lymphocyte reaction, no significant difference in the stimulation index and cytolytic activity was detected between transgenics and control littermates. However, when immunized with a T cell dependent antigen, transgenic mice showed 10-fold higher serum IgG1 titers. This suggests that the expression of transgenic HSA in cells normally negative for HSA (e.g. peripheral T cells or memory B cells) leads to a better stimulation of lymphocytes during a secondary antibody response.
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Alterations in gene expression associated with stepwise acquisition of malignancy in murine cytotoxic T cell lines.
We have isolated a series of variant cell lines from a murine CD8+ T cell clone representing distinct stages in stepwise acquisition of malignancy. A first type of variant has acquired independency of restimulation with MHC/Ag but has kept dependence on IL-2 for continuous growth in culture. A second type of variant has acquired, in addition, independency of IL-2. A third type of variant was isolated from tumors induced upon injection of IL-2 independent variants into syngeneic mice. Clonal relatedness between the cell line was ascertained by Southern blot and sequence analyses of their TCR beta chain genes. The cell lines were analyzed for their expression of genes typical for CD8+ T cells, using Northern blot hybridization, flow cytometry, and functional methods. Concentrating on the transition from IL-2 dependent to IL-2 independent cellular growth, we find the same triad of changes in two independently derived groups of variant cell lines: loss of expression of the CD8 alpha gene with concomitant loss of CD8 from the cell surface, a slight but significant overexpression of IL-2R alpha and beta chains with increased low affinity IL-2 binding sites, and constitutive overexpression of c-myc. Autocrine IL-2 dependent growth could be excluded. Expression of p56lck did not vary between the cell lines. We discuss the possibility that IL-2 independent growth may be associated with intracellular redistribution of p56lck from CD8 alpha to IL-2R beta, thus generating constitutively active IL-2R. Ex vivo established tumor variants differed from their parental culture cell lines by their constitutive secretion of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
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Phenotypic characterization of splenic T cells from mice infected with Plasmodium chabaudi chabaudi.
T cells from spleens of mice infected with the erythrocytic stages of Plasmodium chabaudi chabaudi have been analysed with respect to their expression of surface molecules CD3, CD4 and CD8 and T-cell receptor (TCR) alpha beta and gamma delta. The majority of T cells from infected mice were alpha beta TCR+. However, there was an increase of approximately 8-10-fold in the proportion and total number of gamma delta T cells. Immunocytochemical analysis of sections of spleens taken from infected C57BL/6 mice during a primary infection showed that this increase took place particularly in the non-lymphoid areas. Within the alpha beta TCR+ T-cell population, both CD4+ T cells and CD8+ T cells were represented in proportions similar to those observed in normal uninfected mice. Stimulation of splenic T cells from infected mice with P. chabaudi-infected erythrocytes in vitro resulted in a blasted cell population composed predominantly of alpha beta TCR+ T cells with no preferential expansion of gamma delta TCR+ T cells. There was no evidence of superantigen-like stimulation of T cells bearing particular V beta chains of the TCR. The representation of the different V beta chains within the population was not significantly different from that seen in uninfected mice.
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Autoreactivity of low but not of high CD4 variants of an antigen-specific, I-A-restricted mouse T cell clone.
Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.
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Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes.
The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.
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In vivo depletion of interferon-gamma leads to susceptibility of A/J mice to mouse hepatitis virus 3 infection.
The possible role of interferon-gamma (IFN-gamma) in the resistance of A/J mice to MHV3 infection was investigated. Monoclonal antibodies specific for IFN-gamma, CD4 and CD8 molecules were administered in vivo to deplete selectively the IFN-gamma synthesized or the appropriate subset of T cells. The animals were then infected with MHV3 and the course of infection was followed by studying different parameters, such as, the mortality, the virus growth in the tissues and the IFN-gamma synthesis in sera and peritoneal exudates. After MHV3 infection, a full resistance of control A/J mice was observed, in contrast to the high mortality rate observed among the depleted animals, where higher virus titers were found in different tissues. The IFN-gamma synthesis in sera and peritoneal exudates of depleted mice, after MHV3 infection, drastically decreased when compared to that detected in control mice. The data presented are consistent with the hypothesis that IFN-gamma plays an essential role in the resistance of A/J mice to MHV3 infection.
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Expression of murine soluble CD4 protein in baculovirus infected insect cells.
The expression of murine soluble CD4 (L3T4) protein (sCD4) by baculovirus-infected insect cells was characterized. The yield of sCD4 reached 2 mg/l culture supernatant late in infection. Nevertheless, a large amount of sCD4 remained cell-associated, presumably in the endoplasmic reticulum or an early golgi compartment, as indicated by the endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitivity of its carbohydrate chains. The secreted form of sCD4 is modified with both endo-beta-N-acetyl-D-glucosaminidase D (endo-D) and endo-H-sensitive oligosaccharides. It was possible that the incomplete secretion indicated faulty glycosylation or improper folding of the sCD4 protein. However, inhibitor studies showed that complete carbohydrate processing is not required for secretion of sCD4 by insect cells. Moreover, maintained reactivity with a panel of monoclonal Ab as well as phase partitioning experiments suggested that secretion is apparently not caused by misfolding of the sCD4 protein. Similar results were obtained with biologically active murine interleukin-4 produced by insect cells. This indicates that an inefficient secretory pathway may be a general problem of baculovirus-infected insect cells and is not a consequence of incorrect molecular conformation.
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