H. von Boehmer

Development of the CD4 and CD8 lineage of T cells: instruction versus selection.

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8‐ and CD4–8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up‐regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha‐beta‐) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha‐beta‐ T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.

Surface expression of the beta T cell receptor (TCR) chain in the absence of other TCR or CD3 proteins on immature T cells.

T cell receptor (TCR) beta genes are rearranged prior to TCR alpha genes. A productively rearranged TCR beta gene suppresses further V beta gene rearrangement. Here we show that in beta TCR transgenic mice the TCR beta‐chain can be expressed on the surface of immature CD4–8– thymocytes, but not on mature T cells, in the absence of any other known TCR chain and proteins of the CD3 complex. Analysis by NEPHGE and SDS‐PAGE showed that at least some beta TCR exists on the surface as a large disulfide‐linked complex with unknown acidic molecules. The introduction of the beta TCR gene into scid mice resulted in the expression of the beta TCR on the cell surface of thymocytes and induced the expression of CD4 and CD8 co‐receptors as well as transcription of the alpha TCR locus.

Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

To characterize cis‐acting elements controlling the expression of T cell receptor beta‐chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta‐chain with different extensions of 5′ and 3′ flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements.

Recombinant interleukin 4/BSF-1 promotes growth and differentiation of intrathymic T cell precursors from fetal mice in vitro.

Recombinant mouse interleukin 4/BSF‐1 (rIL4/BSF‐1) together with phorbol myristate acetate (PMA) promotes growth of one out of approximately four intrathymic T cell precursors from fetal mice (14‐15 days gestation). This response is not inhibited by even high concentrations of monoclonal antibody against the receptor for interleukin 2. Fetal thymocytes activated by rIL4/BSF‐1 plus PMA give rise to cytolytic T cells after 7‐21 days of culture. All the proliferating cells are Thy1+, some of them express Lyt2 but none has detectable L3T4 T cell differentiation antigens nor T cell antigen receptor (F23.1) on the cell membrane as assessed by immunofluorescence staining and flow fluorocytometry analysis. It is concluded that rIL4/BSF‐1 exerts both growth and differentiation activities on normal intrathymic T cell precursors. The results provide evidence for an alternative growth factor to interleukin 2 involved in proliferation of T cell precursors. These findings open new and direct ways of studying cellular and molecular events during the differentiation of normal intrathymic T cell precursors in vitro and extend the spectrum of target cells for IL4/BSF‐1.

T Cell Function in Bone Marrow Chimeras; Absence of Host-Reactive T Cells and Cooperation of Helper T Cells across Allogeneic Barriers

Recent observations on tolerance to several antigens, including histocompatibility markers (Droege 1975), suggest active suppression and thus challenge the concept of clona! deletion (Bumet & Fenner 1949, Lederberg 1959). A priori, the concepts of clonal deletion and active suppression are not mutually exclusive: Active suppression might lead to clonal deletion, i.e. to a lack af precursor cells with a specificity for a given antigen. On

Expression of T cell receptors by thymocytes: in situ staining and biochemical analysis

We have examined the in situ expression of T cell receptor (TCR) V beta 8 protein in murine thymus during ontogeny using the monoclonal antibody F23.1. Positive cells were first detected at day 15 of gestation (0.6%). By day 16 the frequency of positive cells increased dramatically (4.18%). From day 16 to day 17 positive cells doubled (8.17%). The first clusters of F23.1 positive cells were seen at day 17. In the cortex, positive cells decreased from 14% in the newborn mice to 9.8% in 8‐week‐old mice, whereas in the medulla the frequency remained unchanged at 20%. The antibody F23.1, as well as an antiserum raised against the constant region of the beta chain, immunoprecipitated receptor dimers from highly purified Lyt2+, L3T4+ thymocytes and from two thymic lymphomas of cortical phenotype which express full size alpha and beta mRNA. The receptor dimer could not be precipitated from Lyt2‐, L3T4‐ thymocytes. The results are discussed with regard to intrathymic T cell repertoire selection.

Selective stimulation by B lymphocytes in the syngeneic mixed lymphocyte reaction

T and B lymphocytes from spleen and thoracic duct have been separated by preparative electrophoresis and their ability to stimulate syngeneic and allogeneic thymic cells in mixed lymphocyte cultures has been studied.

T cell clones: their use for the study of specificity, induction, and effector-function of T cells.

The possibility of studying single B cells, as effector cells in the plaque forming cell (PFC) assay [31], or as PFC-progenitors under conditions of limiting dilution [1, 38], has facilitated the analysis of their specificity, induction, and effector function. Immunoglobulin (IG)-secreting myelomas [-51] and more recently hybridomas [33] are useful tools for biochemical characterization of immunoglobulin and Ig coding genes. Similar tools are needed for the study of T cells. It is the aim of this article to review some of the more recent techniques used to answer some questions about T lymphocytes. These techniques may be grouped into three partly overlapping categories: (1) Induction of T effector cells under conditions of limiting dilution (2) transformation or hybridization of specific T cells and (3) growth and cloning of T cells in media containing T cel ! growth factor(s).

Techniques for Separation and Selection of Antigen Specific Lymphocytes

The immune system consists of a complex array of cells in various stages of differentiation and forms of specialization. Many different subpopulations of lymphocytes have been distinguished according to physical, biochemical, physiologic, and functional properties. There are two major classes of lymphocytes; B cells and T cells. The repertoire of receptors for antigen on mature cells of both classes is very diverse, each individual cell and its progeny being committed and restricted to synthesize molecules with identical antigen-combining sites. This restriction in specificity has been predicted in the clonal selection theory and was first demonstrated at the level of antibody-secreting cells and subsequently also at the level of unstimulated lymphocytes. Techniques which allowed the elimination or isolation of cells expressing specific receptors provided the most convincing experimental evidence for the clonal selection theory.