Hubertus Kohler

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.

ROLE OF POSITIVE AND NEGATIVE CIS-REGULATORY ELEMENTS IN THE TRANSCRIPTIONAL ACTIVATION OF THE LYSOZYME LOCUS IN DEVELOPING MACROPHAGES OF TRANSGENIC MICE

Expression of the chicken lysozyme locus in macrophages is regulated by at least six different positive and negative cis-regulatory elements. Chromatin of the chicken lysozyme locus is gradually reorganized during macrophage differentiation, indicating that each cis-regulatory element is activated at a different developmental stage. Irrespective of their differential developmental activation, individual cis-regulatory regions are capable of driving transcription of the lysozyme gene in mature macrophages of transgenic mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of transcriptional up-regulation of deletion mutants of the lysozyme locus in a new in vitro differentiation system based on enriched primary macrophage precursor cells from the bone marrow of transgenic mice. We show that constructs carrying cis-regulatory elements which are structurally reorganized early in development are also transcriptionally active at an early stage. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. The presence or absence of a negative regulatory element has no influence on the time course of transcriptional activation of the lysozyme locus.

Partial agonism and independent modulation of T cell receptor and CD8 in hapten‐specific cytotoxic T cells

We recently demonstrated antagonism for hapten‐reactive T cells by altered hapten ligands. Here we investigated partial peptide‐ or hapten‐agonism and effects of antigen stimulation on the expression of TCR and the CD8 coreceptor using a set of DNP‐ or TNP‐peptide‐induced, H‐2Kb ‐restricted mouse CTL clones. Various Kb ‐binding TNP‐ and DNP‐peptides acted as partial agonists, cross‐reactively stimulating individual clones for cytotoxicity and IFN‐γ secretion, but failing to induce proliferation or TNF‐α production. Full agonism, i.e. activation of all possible functions, was usually restricted to those hapten‐peptide combinations used for the induction of the respective clones. Our data imply distinctive kinetic optima for TCR antigen contacts in the induction of the various T cell effector functions. Down‐regulation of TCR was efficiently induced by full, but with one exception not by partial, agonists, indicating the independence of cytotoxicity or IFN‐γ secretion from TCR modulation. On the other hand, a reduction of TCR expression induced by full agonists was usually not accompanied by synchronous down‐modulation of CD8 as reported by others for human T cells. In fact, three of four full agonists and all partial agonists markedly enhanced rather than reduced the expression of CD8. Increased CD8 surface levels enhanced cytolytic potential and increased cross‐reactivity patterns of individual clones. Brefeldin A blocked this CD8 induction by partial agonists, and in the case of full agonists resulted in a parallel reduction of both, TCR and CD8. Thus, antigenic stimulation of mouse T cells initially down‐modulates CD8 together with TCR, but the loss of coreceptor is over‐compensated by a signal for increased CD8 export.

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.

Recognition by human V gamma 9/V delta 2 T cells of melanoma cells upon fusion with Daudi cells

Abstract Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level.