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K Eichmann

Publications and source records attributed to K Eichmann.

At least 37 records · Page 2Linked to original sources
PubMed1997

Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells.

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+ population generated a single transient wave of IgM+ IgD+ B cells but failed to generate T cells. In contrast, transfer of the B220- fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.

Animals↗
PubMed1997

Potential immunocompetence of proteolytic fragments produced by proteasomes before evolution of the vertebrate immune system.

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon gamma induction in vivo or addition of recombinant proteasome activator 28alpha in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

Acetylcysteine↗
PubMed1997

Reconstitution of B cell subsets in Rag deficient mice by transplantation of in vitro differentiated embryonic stem cells.

In vitro differentiated embryonic stem (ES) cells contain a population which is similar to fetal liver pro/pre-B cells on the basis of cell surface antigens and cytoplasmic expression of immunoglobin heavy chain. This population was purified and transplanted into Rag-1 deficient recipients to characterize its developmental potential in vivo. Following intravenous transfer, these cells rapidly reconstituted the splenic B but not the T cell compartment. Reconstitution was transient, indicating the lack of long-term reconstituting capacity. Similar to fetal liver, B-1 type as well as conventional B cells were generated, accompanied by high serum IgM levels. Intraperitoneal injection generated high numbers of peritoneal B cells, predominately of the B-1a phenotype, with poor splenic repopulation and low serum IgM levels. These observations suggest the emergence of two different B lineage precursor populations during in vitro ES cell differentiation and define a possible role of the microenvironment in directing lymphoid development.

Animals↗
PubMed1997

Oxidation of N(G)-hydroxyl-L-arginine to nitric oxide mediated by respiratory burst: an alternative pathway to NO synthesis.

N(G)-Hydroxy-L-arginine is an intermediate metabolite in the synthesis of nitric oxide that is upregulated and secreted during acute inflammation in vivo. Previous reports have shown that chemically induced superoxide anion oxidizes N(G)-hydroxy-L-arginine to nitric oxide. Here, we demonstrate that this reaction takes place physiologically in phagocytic cells during the respiratory burst, and is independent of the presence of nitric oxide synthase.

Animals↗
PubMed1997

Asynchronous coreceptor downregulation after positive thymic selection: prolonged maintenance of the double positive state in CD8 lineage differentiation due to sustained biosynthesis of the CD4 coreceptor.

In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream alphabeta T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

Animals↗
PubMed1997

Differentiation of CD4(high)CD8(low) coreceptor-skewed thymocytes into mature CD8 single-positive cells independent of MHC class I recognition.

Thymocytes with a CD4(hi)CD8(lo) coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4(hi)CD8(lo) cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2bm13 background, generate a large number of CD4(hi)CD8(lo) CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4(hi)CD8(lo) population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4(hi)CD8(lo) CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.

Animals↗
PubMed1996

Proliferation kinetics associated with T cell receptor-beta chain selection of fetal murine thymocytes.

After productive rearrangement of a TCR beta chain gene, CD4-8- double negative (DN) thymocytes express TCR beta polypeptide chains on the cell surface together with pre-T alpha and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCR beta + DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas DN cells that fail to generate a productive TCR beta gene rearrangement do not continue in development. This process is termed TCR beta chain selection. Although it is likely that differences between proliferation dynamics of TCR beta + and TCR beta-cells may play a role, the exact mechanisms of TCR beta chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR beta + and TCR beta-thymocytes during fetal development, i.e., when TCR beta chain selection takes place for the first time. We analyzed in situ accumulation of TCR beta + thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR beta + and TCR beta-populations by flow cytometry. About 600 TCR beta + cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5, and 15.5. As of dg 14.5, most TCR beta + cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR beta + cells has slowed down to less than 1/d. About three quarters of TCR beta-cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCR beta-cells, but not TCR beta + cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCR beta-cells. Positive selection of fetal TCR beta + cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.

Animals↗
PubMed1996

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Amino Acid Sequence↗
PubMed1996

Immature T cells in peripheral lymphoid organs of recombinase-activating gene-1/-2-deficient mice. Thymus dependence and responsiveness to anti-CD3 epsilon antibody.

Thymocytes of mice deficient in the recombinase-activating gene (RAG)-1 or RAG-2 cannot express and receive signals through the pre-TCR. As a result, thymocyte development in these mice terminates at the CD4/8 double negative (DN), IL-2R-alpha-positive stage. Nevertheless, RAG-deficient DN thymocytes express functional CD3 complexes and can therefore be induced by anti-CD3 epsilon mAb to mature to the CD4+8+ double positive stage. In the present paper we demonstrate that the peripheral lymphoid organs (lymph nodes, spleen) and peripheral blood of RAG-deficient mice harbor an immature T cell population which, similar to RAG-deficient DN thymocytes, contains high levels of cytoplasmic CD3 epsilon and responds to anti-CD3 epsilon mAb in vivo. With respect to surface phenotype (Thy1.2+, PgP-1+, HSA+, Fc gamma RII/III-, IL-2R-alpha-, c-kit-), these cells are similar to intermediate stage RAG-deficient DN thymocytes. Moreover, they express mRNA for pre-TCR-alpha and for the nondeleted RAG. Following injection of anti-CD3 epsilon mAb, these cells proliferate, down-regulate heat stable Ag and PgP-1, and partially differentiate to CD4+ and CD8+ double positive and single positive cells. The induced population displays a mixed phenotype, between that of immature thymocytes and lymph node T cells in normal mice. Induction is successful in thymectomized RAG-deficient mice, suggesting that it occurs in the periphery. However, after thymectomy, inducible cells disappear with an approximate half-life of 10 to 14 days. We suggest that DN thymocytes can emigrate and repopulate peripheral lymphoid organs of RAG-deficient mice. These cells respond to CD3 signaling by aberrant maturation, possibly due to the inappropriate microenvironment of peripheral lymphoid organs.

Animals↗
PubMed1996

A novel mouse thymocyte antigen (F3Ag): down-regulation during the CD4+CD8+ double-positive stage indicates positive selection.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.

Animals↗
PubMed1995

Transcription of granzyme A and B genes is differentially regulated during lymphoid ontogeny.

During development, thymocytes express a number of genes typical for activated peripheral T lymphocytes, including granzymes. We have now analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and cytochemistry fetal liver cells and thymocytes at various developmental stages for the expression of granzyme A-G genes. At days 13-17 of gestation, only granzyme B but none of the other granzymes is expressed in fetal liver. In the most immature, Pgp-1+IL2R alpha-, thymocyte subpopulation mRNAs for granzymes A-C but not for granzymes D-G are detectable. Upon further differentiation via Pgp-1-IL-2R alpha + into more mature Pgp-1-IL-2R alpha- thymocytes the level of expression of granzymes A, B, and C gradually declines reaching its lowest level at the CD4+ 8+ double positive stage. In fetal thymic lobes depleted of lymphoid cells by treatment with deoxyguanosine, no transcripts for granzymes A, B, and C were found indicating that the PCR signals are derived exclusively from early precursor T/natural killer (NK) lineage cells rather than from residual stromal elements. In mature CD4+CD8- and CD4-CD8+ thymocytes, granzyme B mRNA is found at similar levels in both subsets whereas granzyme A mRNA is expressed selectively in the CD4-CD8+ subset. Enzymatic activity of granzyme A was only seen in a fraction of CD4-CD8+ thymocytes negative for heat stable antigen (HSA) but not in the more immature HSA+ fraction of CD4-CD8+ thymocytes. The data suggest that (a) granzyme B is a pro-thymocyte marker for all T/NK lineage cells; (b) granzyme A transcripts are associated with thymocytes with the potential to develop into the CD8+ lineage; and (c) granzyme A enzymatic activity is only expressed in the most mature CD4-CD8+ stage, suggesting that granzyme proteins are not involved in early stages of thymocyte development.

Animals↗
PubMed1995

Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages.

The present study addresses the regulatory mechanisms involved in the arginine metabolism of macrophages by arginase and nitric oxide synthase. Induction of both enzymes with LPS or by mixed lymphocyte reaction has been reported. Here, we demonstrate that these enzymes can be independently induced in murine bone-marrow-derived macrophages with the appropriate agonists. Arginase expression is specifically triggered by IL-4, IL-10, PGE2 as well as non-toxic or detoxified LPS. Conversely, IFN gamma induces only NO synthesis in these cells. The results demonstrate that the metabolism of arginine in macrophages is controlled by TH1/TH2-dependent cytokines and suggest a regulatory role of arginase on the NO synthesis by intracellular substrate depletion.

Amino Acid Oxidoreductases↗
PubMed1995

Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines.

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively.

Amino Acid Oxidoreductases↗
PubMed1995

Regulation of T cell receptor (TCR)-beta locus allelic exclusion and initiation of TCR-alpha locus rearrangement in immature thymocytes by signaling through the CD3 complex.

During thymocyte differentiation, the T cell receptor (TCR)-beta genes are rearranged before the TCR-alpha genes. Immature CD4-8- double-negative thymocytes with a productive rearrangement of the TCR-beta locus are selected to continue maturation to the CD4+8+ double-positive stage, driven by signals through the pre-TCR. The signals through the pre-TCR can be synchronized by injection of mice with anti-CD3 epsilon monoclonal antibody. Using this approach, we demonstrated coordinated induction of a triad of responses in immature thymocytes: arrest of V to DJ rearrangement in the TCR-beta locus, transient down-regulation of rearrangement-activating gene (RAG)-1 and RAG-2 transcripts, and initiation of germ-line transcription of the TCR-alpha locus. These results suggest that the transition from TCR-beta to TCR-alpha locus rearrangement is controlled by signal transduction through the pre-TCR.

Alleles↗
PubMed1995

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR beta locus is incomplete but thymocyte development is not restored by TcR beta or TcR alpha beta transgenes.

The protein tyrosine kinase, p56lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56lck expression (p56lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4+CD8+ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56lck-/- mice and found an increase in the proportion of the CD44-CD25+ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) beta expression, may be delayed in the absence of p56lck expression. In addition, the expression of a transgenic TcR beta chain or TcR alpha beta pair did not restore thymic development in p56lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one V beta chain in TcR transgenic p56lck-/- mice, we found that inhibition of endogenous TcR beta gene rearrangement was almost complete in thymocytes of V beta transgenic p56lck-/- mice and we could not detect any peripheral T cells that expressed more than one V beta chain in non-transgenic p56lck-/- mice.

Alleles↗
PubMed1995

A signal strength hypothesis of thymic selection: preliminary considerations.

During their differentiation, thymocytes are subjected to two rounds of selection. First, CD4-8- double-negative (DN) thymocytes with a functional TCR-beta chain express a alpha-beta+ CD3 complex on their surface and, as a consequence, are selected to mature to the CD4+8+ double-positive (DP) stage. This round ends after the initial proliferation of young DP thymocytes and is termed beta-chain selection. Second, DP thymocytes are selected on the basis of their alpha+beta+ CD3 complex. This is termed repertoire selection and the cells are given three choices: death by neglect selection, death by positive selection, or deletion by negative selection. Using anti-CD3 epsilon mAb as invariant ligand, signals for beta-chain selection of DN cells including proliferation of DP cells do not require a Ca2+ response, are independent of CD3 zeta, and are only slightly impaired in the absence of p56lck (lck). Signals that induce positive selection of DP thymocytes require a partial Ca2+ response and CD3 zeta but are independent of lck. Deletion of DP thymocytes requires a full-blown Ca2+ response and both, CD3 zeta and lck. Thymic selection thus appears to be governed by a gradient of signal intensities.

CD3 Complex↗
PubMed1995

Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules.

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

Amino Acid Sequence↗