Pawel Kisielow

The thymus selects the useful, neglects the useless and destroys the harmful

Although efficient at reacting to foreign antigen in the context of MHC, mature T cells do not normally react to self antigens presented by self MHC. In this review, Harald von Boehmer and colleagues describe the investigation of self MHC restriction and self-tolerance using TCR transgenic mice expressing a receptor for the male-specific minor histocompatibility antigen, H-Y, in the context of class I H-2Db MHC antigens, on many of their T cells. CD4-8+ T cells expressing the transgenic receptor were positively selected by the restricting H-2Db MHC antigens in female transgenic mice. In the male TCR transgenic mice, CD4+8+ thymocytes were deleted, and transgene-expressing T cells with high surface-density of CD8 were-absent from the periphery. The remaining T cells could not be activated by male H-Y stimulator cells, as they lacked or expressed only low levels of CD8 molecules.

Detection of apoptosis of immature CD4+8+ thymocytes by flow cytometry

Apoptosis, i.e., programmed cell death, may be the mechanism by which both autoreactive and unselected immature CD4+8+ thymocytes are eliminated in the thymus. In the present paper we describe a simple and rapid flow cytometric method which permits one to study the induction and kinetics of apoptosis of CD4+8+ thymocytes using in vivo and in vitro suspension cultures. Analysing the level of surface expression of CD4 and CD8 molecules, forward light scatter and side (90 degrees) scatter as well as staining with ethidium bromide, three distinct stages of apoptosis of CD4+8+ thymocytes were defined. By counting cells passing through different stages of apoptosis one can attempt to quantitate this process. This method should be useful for in vitro studies on the mechanisms of negative and positive selection of CD4+8+ thymocytes, i.e., induction and inhibition of apoptosis respectively.

Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4+ Foxp3+ regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4+ Foxp3− T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4+ T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4+ Foxp3+ and CD4+ Foxp3− T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice.

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic αβ heterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenic α chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both α and β transchains and virtually all fetal thymocytes expressed the transgenicαβ heterodimer by day 17. The early expression of the transgenic TCR on CD4-8- thymocytes prevented the development of γδ cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8- thymocytes than the female transgenic thymus. The preponderance of CD4-8- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+ thymocytes and their CD4-8+ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16. The preferential accumulation of CD4-8+ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was .obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+ thymocytes to the CD4-8+ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+ thymocytes into CD4-8+ T cells. In normal mice, the precursors of CD+4+8 and single positive thymocytes have the CD4-8- CD3-J11d+ (or M1/69 +) phenotype. Because of the early expression of the transgenic αβ heterodimer, this population was not detected in adult transgenic mice. All CD4-8- M1/ 69+ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.

Lymphocyte lineage commitment: Instruction versus selection

In mammals, lymphocytes develop from hematopoietic stem cells throughout life. The exact branchpoint of irreversible commitment to the lymphocyte lineage is not known, and developmental stages committed to the T and B lymphocyte lineages are being defined. The T cell lineage branches further into cells with ~8 or a8 T cell receptors (TCRs) for antigen. The a8 TCR lineage is subdivided by surface markers and functional potential into CD4+8helper and CD4-8+ killer lineages, which are discussed here. CD4+ He/per and CDl Connolly et al., 1988): cross-linking of either a CD4 or CD8 coreceptor with the a8 TCR by the same MHC molecule is required for efficient activation of T cells (Emmrich et al., 1986; Gabert et al., 1987). For some time, it has not been clear whether CD4+ and CD8’ T cells can each express both class I and class II MHC-specific TCRs. If they can, those cells with a “mismatched” combination of coreceptor and a6 TCR (which would bind to different MHC molecules) would be wasted because these cells could not be efficiently activated. The alternative possibility is that the formation of the CD4+ helper and CD8+ killer lineages is crucially dependent on the specificity of the a8 TCR for class II and class I MHC molecules, respectively. To probe the latter idea, the contribution of a particular receptor (of defined antigen specificity) to the formation of CD4 and CD8 lineages needed to be examined. Because of the great TCR diversity, this could not be done in normal mice but required TCR transgenic mice. The results of the transgenic experiments showed, first, that binding of an up TCR to the polymorphic part of thymic MHC molecules in the absence of the specific peptide antigen is essential for the generation of functional CD4+ helper and CD8+ killer lineage cells from nonfunctional CD4+8+ intrathymic precursors. Second, a CISSS l-specific TCR exclusively supports the development of mature CD8+, while a class II-specific TCR exclusively supports the development of mature CD4+ lineage Cells (von Boehmer, 1990). These differentiation Steps are called positive selection. Minireview

Negative selection of the T‐cell repertoire: where and when does it occur?

Summary:  Because of the use of somewhat artificial models for the elucidation of negative selection [superantigen, T‐cell receptor (TCR) transgenic mice], there is still considerable uncertainty at what stages of T‐cell development negative selection can occur and whether it becomes manifest as developmental arrest, lineage diversion, or induction of apoptotic cell death. Here, experimental evidence is reviewed that excludes developmental arrest and lineage diversion as the sole mechanisms of negative selection. The data emphasize that both CD4+CD8+ double‐positive cortical as well as semi‐mature, single‐positive, medullary thymocytes are targets of deletion in experimental models employing superantigen and TCR transgenic mice with premature as well as ‘timely’ onset of TCR expression.

Differences in Expression Level of Helios and Neuropilin-1 Do Not Distinguish Thymus-Derived from Extrathymically-Induced CD4+Foxp3+ Regulatory T Cells

Helios transcription factor and semaphorin receptor Nrp-1 were originally described as constitutively expressed at high levels on CD4+Foxp3+ T regulatory cells of intrathymic origin (tTregs). On the other hand, CD4+Foxp3+ Tregs generated in the periphery (pTregs) or induced ex vivo (iTregs) were reported to express low levels of Helios and Nrp-1. Soon afterwards the reliability of Nrp-1 and Helios as markers discriminating between tTregs and pTregs was questioned and until now no consensus has been reached. Here, we used several genetically modified mouse strains that favor pTregs or tTregs formation and analyzed the TCR repertoire of these cells. We found that Tregs with variable levels of Nrp-1 and Helios were abundant in mice with compromised ability to support natural differentiation of tTregs or pTregs. We also report that TCR repertoires of Treg clones expressing high or low levels of Nrp-1 or Helios are similar and more alike repertoire of CD4+Foxp3+ than repertoire of CD4+Foxp3- thymocytes. These results show that high vs. low expression of Nrp-1 or Helios does not unequivocally identify Treg clones of thymic or peripheral origin.

The Expression of CD4 and CD8 Accessory Molecules on Mature T Cells is not Random but Correlates with the Specificity of the αβ Receptor for Antigen

The expression of CD4 or CD8 molecules was initially believed to correlate with lymphocytes exhibiting distinct effector functions, i.e. helper and killer T lymphocytes (Kisielow et al. 1975, Cantor & Boyse 1975, Dialynas et al. 1983). When it became apparent that the effector functions of CD4 and CD8 lymphocytes were not as distinct as initially believed, i.e. CD4 cells could become cytotoxic (Krensky et al. 1982, Bell & Stastny 1982) and CD8 cells could produce at least some lymphokines (von Boehmer & Haas 1981, Widmer & Bach 1981), it was pointed out that the CD4 and CD8 accessory molecules may correlate with T-cell specificity, i.e. MHC restriction rather than lymphocyte function (Swain 1983). Even though there are apparent exceptions to this rule we think that there is a true correlation between the specificity of the ajff T-cell receptor and the expression of CD4 or CD8 molecules by mature T cells (Haas & von Boehmer 1984). We believe that the apparent exceptions simply reflect fortiiitous cross reactions of self MHC-restricted afi receptors on T cells which are selected by thymic MHC antigens to be either class I or class II MHC-restricted (Haas & von Boehmer 1984, Teh et al. 1988). Some time ago, we postulated that during

Identification of a third evolutionarily conserved gene within the RAG locus and its RAG1-dependent and -independent regulation.

Recombination‐activating gene (RAG)1 and RAG2 encode T and B lymphocyte‐specific endonucleases indispensable for rearrangements of antigen‐receptor gene segments but also capable of causing deleterious chromosome rearrangements. The mechanisms regulating RAG expression and repression are not clear. Here we identify NWC, a third evolutionarily conserved gene within the RAG locus, and show that it is ubiquitously expressed, with the notable exception of RAG‐nonexpressing immature and mature T and B lymphocytes because in lymphocytes it is regulated by the RAG1 promoter and transcribed as RAG1–NWC hybrid mRNA molecules. We also show that in all other cells NWC is controlled by the RAG2 intragenic promoter, which in immature and mature T and B lymphocytes is silent. The possible implications of these findings for understanding the activation and inactivation of RAG genes in lymphocytes and their repression in other cells are discussed.

Inhibition of MEK induces fas expression and apoptosis in lymphomas overexpressing Ras.

Published results implicate PI3 kinase as a target of oncogenic Ras activity leading to the suppression of Fas but whether other Ras targets (e.g. Raf-1) are also involved is unclear. Here we report that thymic lymphomas overexpressing Ras and Raf-1 exhibit low expression of Fas. We show that expression of Fas in these lymphomas can be increased not only in the presence of a specific inhibitor (LY294002) of PI3 kinase, but also in the presence of specific inhibitor (PD98059) of MEK, downstream target of Raf-1. Both treatments result in accumulation of ERK in cytosol of lymphoma cells suggesting cross-talk between these two pathways regulating Fas expression. Treatment with PD98059 also results in apoptosis of the lymphoma cells but not of normal thymocytes expressing low Raf-1 levels. These observations provide evidence for involvement of Raf-1/MEK/ERK pathway in Ras-mediated inhibition of Fas expression and in selective promotion of survival of lymphoma cells.