Klaus Eichmann

The Duration of Nuclear Residence of NFAT Determines the Pattern of Cytokine Expression in Human SCID T Cells

The expression of cytokine genes and other inducible genes is crucially dependent on the pattern and duration of signal transduction events that activate transcription factor binding to DNA. Two infant patients with SCID and a severe defect in T cell activation displayed an aberrant regulation of the transcription factor NFAT. Whereas the expression levels of the NFAT family members NFAT1, -2, and -4 were normal in the patients’ T cells, dephosphorylation and nuclear translocation of these NFAT proteins occurred very transiently and incompletely upon stimulation. Only after inhibition of nuclear export with leptomycin B were we able to demonstrate a modest degree of nuclear translocation in the patients’ T cells. This transient activation of NFAT was not sufficient to induce the expression of several cytokines, including IL-2, IL-3, IL-4, and IFN-γ, whereas mRNA levels for macrophage inflammatory protein-1α, GM-CSF, and IL-13 were only moderately reduced. By limiting the time of NFAT activation in normal control cells using the calcineurin inhibitor cyclosporin A, we were able to mimic the cytokine expression pattern in SCID T cells, suggesting that the expression of different cytokine genes is differentially regulated by the duration of NFAT residence in the nucleus.

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.

Receptors and signals in early thymic selection

During differentiation, thymocytes are selected twice for expressing a functional version of the CD3-T cell receptor (TCR). A first round of selection involves the immature CD4CD6double-negative (DN) ceils that express interleukin-2 receptor-a (IL-2Ra). At this stage, thymocytes rearrange their TCRj3 genes in a random fashion (Godfrey et al., 1993) leading to a successful rearrangement in approximately 5 of 9 cells (i.e., 56%). Thymocytes that productively rearranged the TCR8 locus are selected by virtue of expressing an immature form of the CD3-TCR, termed pre-TCR, to proliferate, down-regulate IL-2Ra and mature to the CD4+CD8+ double-positive (DP) stage (Groettrup and von Boehmer, 1993; Levelt and Eichmann, 1993). At the same time, TCR8 locus rearrangement is arrested to achieve allelic exclusion. A second round of selection takes place during the DP stage. At this stage, the TCRa genes are rearranged (Petrie et al., 1993) and thymocytes are subsequently subjected to repertoire selection based on the specificity of the mature a+j3+CD3 TCR (Kisielow et al., 1988). DP thymocytes that are positively selected develop into mature CD4+ or CD8+ singlepositive (SP) thymocytes (Chan et al., 1993; Davis et al., 1993). This review deals with the first round of selection in Tcell development: selection for functional rearrangement and expression of the TCR6 chain. The important role of the TCR8 chain in thymic development has been brought out first by studies on mice carrying a functionally rearranged TCR8 transgene (von Boehmer, 1990). In normal mice, the TCR8 transgene arrested the rearrangement of the endogenous TCRj3 gene loci, reflecting the process of allelic exclusion (Uematsu et al., 1988). In Scid mice (Schuler et al., 1986) in which thymocyte development is arrested at the DN IL-2Ra+ stage (Habu et al., 1987; Shoreset al., 1990) aTCR5 transgene restored maturation of thymocytes to the DP stage (von Boehmer, 1990). The pivotal role of the TCR8 chain in early T cell development was further corroborated by the phenotypes of several mutant mouse strains: In mice that cannot produce a TCR8 chain because of a deficiency in the rearrangement machinery (RAG7or RAG2deficient mice) (Mombaerts et al., 1992a; Shinkai et al., 1992a) or because of a mutation in the TCRj3 chain itself (Mombaerts et al., 1992b), thymocyte development is blocked at the DN stage. Introduction of aTCR8 transgene in any of these mice restores T cell maturation to the DP stage (Mombaerts et al., 1992b; Shinkai et al., 1992b). In contrast with TCR8deficient mice, the thymus of TCRa-deficient mice contains normal numbers of DP thymocytes (Philpott et Review

The specificity of proteasomes: impact on MHC class I processing and presentation of antigens

Summary: We have studied polypeptide processing by purified proteasomes, with regard to proteolytic specificity and cytotoxic T‐lymphocyte (CTL) epitope generation. Owing to defined preferences with respect to cleavage sites and fragment length, proteasomes degrade polypeptide substrates into cohorts of overlapping oligopeptides. Many of the proteolytic fragmetits exhibit structural features in common with major histocompatibility complex (MHC) class I ligands including fragment size and frequencies of amitio acids at fragment boundaries. Proteasomes frequently generate definitive MHC class I ligands and/or slightly longer peptides, while substantially larger peptides are rare. Individual CTL epitopes are produced in widely varying amounts, often consistent with immunohier‐archies among CTL epitopes. We further found that polypeptide processing is remarkably conserved among proteasomes of enkaryotic origin and that invertebrate proteasomes can efficiently produce known high‐copy MHC class I ligands, suggesting evolutionary adaptation of the transporter associated with antigen processing and MHC class I to ancient constraints imposed by proteasomal protein’ degradation.

The Response of CD4+ T Cells to Plasmodium chabaudi chabaudi

We have studied the role of CD4+ T cells in the immune response to Plasmodium chabaudi chabaudi. From in vivo experiments in which the different subsets of T cells were depleted, it is clear that CD4+ T cells are essential for the generation of protective immunity. Our limiting dilution analysis show that the CD4 T-cell response to P. chabaudi antigens is heterogeneous, in that distinct functions can be performed by different responding T cells, and these responses change during infection. During the first phase of the infection the predominant response is that of a TH1-type cell, producing IL-2 and IFN-gamma. This correlates with the appearance of IFN-gamma in the serum of infected animals. After the clearance of the acute parasitemia, i.e. in the second phase of the infection, the specific response is characterised by TH2 cells, which are effective helper cells for antibody production and presumably are necessary for the switch of IgM to IgG. CD4+ T cells are effector cells are not necessary in the second phase of the infection; mice which have been depleted of CD4+ T cells at this time are able to control their infection in a manner similar to untreated mice. This ability to control parasitemia coincides with the production of specific IgG but not IgM antibodies and the predominance of TH2 type helper cells. Therefore, our data suggest that malaria-specific IgG antibodies are important effectors in the second phase of an infection with P. chabaudi chabaudi.

Regulation of thymocyte development through CD3: Functional dissociation between p56lck and CD3ζ 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.

Expression of housekeeping and immunoproteasome subunit genes is differentially regulated in positively and negatively selecting thymic stroma subsets.

The expression of housekeeping and/or immunoproteasomes in isolated thymic stroma subsets has so far not been analyzed but may have important consequences for self peptide repertoires presented by MHC class I molecules during positive and negative thymic selection. Here we determined the expression of housekeeping and immunoproteasome β subunits and of PA28 in positively and negatively selecting stroma subsets. Positively selecting cortical thymic epithelial cells (cTEC) expressed only housekeeping but no immunoproteasome β subunit mRNA and proteins. However, immunoproteasome β subunits could be induced in cTEC by infection with Listeria monocytogenes or injection of IFN‐γ. In negatively selecting stroma including medullary epithelial cells and dendritic cells, incomplete and low representation of housekeeping β subunit proteins but high and complete expression of immunoproteasome β subunit proteins suggests absence of proper housekeeping proteasomes and predominance of immunoproteasomes. Expression of immunoproteasome β subunits in negatively selecting stroma was independent of IFN‐γ receptor as shown in knockout (KO) mice. Absence of LMP2 altered thymic selection of the MHC class I‐restricted transgenic P14 TCR in KO mice. The data suggest that negative selection may primarily involve immunoproteasome peptide repertoires and that peripheral infection may influence peptide repertoires involved in positive selection.

Network regulation among T cells: qualitative and quantitative studies on suppression in the non-immune state.

Whereas most theories on the function of the immune system are primarily concerned with an understanding of the immune response to antigen, the network concept (Jerne 1974a, 1974b, Hoffmann 1975, Bona 1981, Lindemann 1979, Urbain et al. 1981) mainly aims at a description of the steady states postulated to exist in the notinal immune system. Theories that are concerned with the immune response usually take the existence of a steady state in the normal immune system for granted, thus ignoring basic questions such as how the system maintains its overall cell numbers when, e.g., in the mouse, greater than 10̂ lymphocytes are born (Metcalf 1967, Jerne 1971), and an unknown number of lymphocytes die each day with no obvious mechanism at work to co-ordinate birth and death rates. More interestingly, how does the system, without antigenic stimulation, maintain a given number of cells/clone in order to provide sufficient lymphocytes for responses to any of a very large number of different antigens? Answers to such questions may perhaps facilitate the understanding of the more complicated mechanisms of response. How does one analyse the normal immune system? While resting lymphocytes are not easily analysed in functional assays, analyses of lymphocytes after stimulation with antigen will provide infonnation only on the portion of the immune system that reacted to the antigen. We have, therefore, decided to analyse lymphocytes after polyclonal stimulation. Such experiments are based on the philosophy that the polyclonally stimulated lymphocyte population is, with respect to the frequency of each cell, similar to the normal lymphocyte population (Andersson et al. 1977, Eichmann et al. 1983). In addition, all cells are in a similar state of activation and can be functionally analysed. We are aware of the problems

Differential proteasomal processing of hydrophobic and hydrophilic protein regions: Contribution to cytotoxic T lymphocyte epitope clustering in HIV-1-Nef

HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and/or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions.

Analysis of direct and cross-presentation of antigens in TPPII knockout mice.

Tripeptidyl peptidase II (TPPII) is an oligopeptidase forming giant complexes in the cytosol that have high exo-, but also, endoproteolytic activity. Immunohistochemically, the complexes appear as distinct foci in the cytosol. In part controversial biochemical and functional studies have suggested that TPPII contributes, on the one hand, positively to Ag processing by generating epitope carboxyl termini or by trimming epitope precursors, and, on the other, negatively by destroying potentially antigenic peptides. To clarify which of these roles is predominant, we generated and analyzed TPPII-deficient mice. Cell surface levels of MHC class I peptide complexes tended to be increased on most cell types of these mice. Although presentation of three individual epitopes derived from lymphocytic choriomeningitis virus was not elevated on TPPII−/− cells, that of the immunodominant OVA epitope SIINFEKL was significantly enhanced. Consistent with this, degradation of a synthetic peptide corresponding to the OVA epitope and of another corresponding to a precursor thereof, both being proteasomally generated OVA fragments, was delayed in TPPII-deficient cytosolic extracts. In addition, dendritic cell cross-presentation of phagocytosed OVA and of OVA internalized as an immune complex was increased to about the same level as direct presentation of the Ag. The data suggest a moderate, predominantly destructive role of TPPII in class I Ag processing, in line with our finding that TPPII is not induced by IFN-γ, which up-regulates numerous, predominantly constructive components of the Ag processing and presentation machinery.