Programmed death of autoreactive thymocytes

T lymphocytes bearing high-affinity T-cell receptors (TCR) for self-antigens are clonally deleted during thymus development. Several recent studies have identified variable domains of the beta-chain of the TCR that are specifically deleted in vivo in mouse strains that express major histocompatibility complex class II molecules in addition to poorly defined self-antigens, including those encoded by the Mls-1a and Mls-2a loci. Deletion of autoreactive cells in these systems occurs in the thymus, and antibody blocking experiments in vivo have implicated the phenotypically immature CD4+CD8+ 'cortical' subset as the target population for clonal deletion. Similarly, studies with transgenic mice bearing autoreactive TCR have provided independent evidence that clonal deletion occurs at the CD4+CD8+ stage of development. But none of these studies directly identified dying autoreactive cells, and the circumstances leading to deletion remain unclear. Here we report that neonatal thymus contains a significant population of phenotypically mature CD4+CD8- cells bearing autoreactive TCR. When placed in short-term culture, a large proportion (60%) of these autoreactive cells die selectively. Furthermore, their death can be prevented by inhibitors of macromolecule (RNA and protein) synthesis, as is the case for glucocorticoid-induced death of thymocytes. These data indicate that physiological clonal deletion of autoreactive cells involves 'programmed' cell death, and that it can occur in cells with a mature (CD4+CD8-) surface phenotype.     

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes

One mechanism by which the immune system develops the ability to discriminate self from nonself is the deletion of autoreactive T-cell clones during thymic maturation. The drug cyclosporin A (CsA) has been shown to interfere with this process, allowing the escape of normally 'forbidden' T-cell clones and the appearance of autoimmune disease. Recently, it has been demonstrated that immature thymocytes undergo programmed cell death (apoptosis) upon activation via the T-cell receptor. A similar phenomenon of activation-induced cell death (AICD) has been observed in T-cell hybridomas. Here we show that AICD in T-cell hybridomas in vitro and in thymocytes in vivo is blocked by CsA. Thus, clonal deletion may involve AICD when self-reactive, immature T cells are induced by self antigen, and CsA may cause autoimmunity by interfering with this process.     

Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis

Antigen receptor stimulation of mature alpha beta T lymphocytes can lead either to proliferation or death. Programmed cell death, termed apoptosis, leads to the clonal deletion of both thymocytes and mature T cells that establishes tolerance. How a mature T cell selects between proliferation and death is not understood. Here I show that interleukin-2 (IL-2) is a critical determinant of the choice between these two fates. Both CD4+ and CD8+ T cells previously exposed to IL-2 undergo apoptosis after antigen-receptor stimulation. Antibody blockade of IL-2 but not IL-4 reverses the marked reduction of lymph node V beta 8+ T cells caused in mice by the bacterial superantigen Staphylococcus aureus enterotoxin B. IL-2 may thus participate in a feedback regulatory mechanism by predisposing mature T lymphocytes to apoptosis.     

Characteristics of TCRαβ+CD4-CD8- thymocytes in fetal thymic organ culture

TCRαβ+CD4-CD8- (TCR+ DN) thymocytes at different developmental periods, i.e. after either 9 or 18 days of culture in the fetal thymic organ culture (FTOC) system, were characterized in the properties of phenotype, proliferation, differentiation and apoptosis. The results showed that anti-CD3 mAb significantly promoted proliferation of TCRαβ+ DN cells generated after 18 days of culture in FTOC, whereas the cells generated after 9 days of culture responded to anti-CD3 mAb by proliferation weakly. IL-7 efficiently induced TCRαβ+ DN cells at day 9 of FTOC to differentiate into TCRαβ+CD4+/CD8+ SP cells without detectable transitional stage of TCRαβ+CD4+CD8+ (DP) cells. In contrast, fewer TCRαβ+ DN cells generated after 18 days of FTOC were induced to differentiate into SP cells. The thymic stromal cell line MTEC5 cells synergized with IL-7 to promote the differentiation of TCRαβ+ DN cells. In addition, TCRαβ+ DN cells were shown to be less susceptible to apoptosis compared with the other major thymocyte subsets. Taken together, these data have provided insight into the characteristics of TCRαβ+ DN thymocytes.     

Negative selection of murine medullary-type single positive thymocytes

Negative selection depletes self-reactive T cells, thus ensuring self-tolerance. It is usually considered that negative selection imposed on double-positive (DP) thymocytes that reside at the cortico-medullary junction. Negative selection model was set up by injecting mice with anti-T cell receptor (TCR) monoclonal antibody (mAb) intraperitoneally in this work. As shown in phenotypic analysis of thymocytes, negative selection destroys not only cortical-type DP thymocytes, but also medullary-type CD3+TCRαβ+CD4SP and CD3+TCRαβ+CD8SP thymocytes. Negative selection of medullary-type single positive (SP) are more susceptible to apoptosis, while with development of the cells, their resistance to apoptosis increases. Therefore, negative selection does not operate on functionally mature thymocytes at the late stage. This result is a supplement to the traditional theory of negative selection. Negative selection of medullary-type thymocytes is probably to further deplete self-reactive T cells, thus producing precise TCR repertoire and inducing self-tolerance.     

Inhibitory Effect of Lithium Chloride on Mouse Thymocyte Apoptosis

This study investigates the effect of lithium chloride (LiCI) on mouse thymocyte apoptosis. A primary culture of mouse thymocytes was preincubated with LiCI (from 5 to 500μmol/L) before exposure to dexamethasone (DEX), the apoptosis inducer. With 100μmol/L of LiCI, apoptotic cell death induced by DEX was almost completely prevented as determined by flow cytometric analysis, terminal deoxynudeotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and DNA laddering assay. The results show that the DEX-induced increment of caspase-3 activity in thymocytes is completelye liminated by LiCI preincubation. The results suggest that LiCI may protect Balb/c mouse thymocytes from apoptosis induced by glucocorticoid in a dose-dependent matter.     

Dampening of death pathways by schnurri-2 is essential for T-cell development

Generation of a diverse and self-tolerant T-cell repertoire requires appropriate interpretation of T-cell antigen receptor (TCR) signals by CD4(+?) CD8(+) double-positive thymocytes. Thymocyte cell fate is dictated by the nature of TCR-major-histocompatibility-complex (MHC)-peptide interactions, with signals of higher strength leading to death (negative selection) and signals of intermediate strength leading to differentiation (positive selection). Molecules that regulate T-cell development by modulating TCR signal strength have been described but components that specifically define the boundaries between positive and negative selection remain unknown. Here we show in mice that repression of TCR-induced death pathways is critical for proper interpretation of positive selecting signals in vivo, and identify schnurri-2 (Shn2; also known as Hivep2) as a crucial death dampener. Our results indicate that Shn2(-/-) double-positive thymocytes inappropriately undergo negative selection in response to positive selecting signals, thus leading to disrupted T-cell development. Shn2(-/-) double-positive thymocytes are more sensitive to TCR-induced death in vitro and die in response to positive selection interactions in vivo. However, Shn2-deficient thymocytes can be positively selected when TCR-induced death is genetically ablated. Shn2 levels increase after TCR stimulation, indicating that integration of multiple TCR-MHC-peptide interactions may fine-tune the death threshold. Mechanistically, Shn2 functions downstream of TCR proximal signalling compenents to dampen Bax activation and the mitochondrial death pathway. Our findings uncover a critical regulator of T-cell development that controls the balance between death and differentiation.     

Adenovirus-mediated<Emphasis Type="Italic">CTLA4-FasL</Emphasis> gene transfer prevents autoimmune diabetes in mice induced by multiple low doses of streptozotocin

Type 1 diabetes is the result of a selective destruction of insulin-producing β cells in pancreatic islets by autoreactive T cells. Depletion of autoreactive T cell through apoptosis may be a potential strategy for the prevention of autoimmune diabetes. Simultaneous stimulation of Fas-mediated pathway and blockade of costimulation by a CTLA4-FasL fusion protein has been reported to lead to substantial inhibition of mixed lymphocyte reaction and enhanced in vitro apoptosis of peripheral lymphocytes. To test the feasibility of CTLA4-FasL-based gene therapy to prevent autoimmune diabetes, we developed recombinant adenovirus containing human CTLA4-FasL gene (AdCTLA4-FasL). A single injection of 2×10~8 plaque forming units (PFU) of AdCTLA4-FasL via tail vein dramatically reduced the incidence of autoimmune diabetes in mice induced by multiple low doses of streptozotocin. AdCTLA4-FasL administration maintained islet insulin content, significantly increased apoptosis of pancreatic lymphocytes, quantitatively     

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.     

Major histocompatibility complex-linked specificity of gamma delta receptor-bearing T lymphocytes

Several recent studies have identified a distinct subset of CD3(T3)+CD4-CD8-T lymphocytes that express a CD3-associated heterodimer made up of the protein encoded by the T-cell receptor (TCR) gamma-gene and a second glycoprotein termed TCR delta (refs 1-4). TCR gamma delta is expressed on CD3+ thymocytes during fetal ontogeny before the appearance of TCR alpha-beta (alpha beta) (refs 5-7), on CD3+CD4-CD8- adult thymocytes, and on a subset (1-10%) of CD3+ cells in adult peripheral lymphoid organs and the peripheral blood. TCR gamma delta-expressing T cells probably represent a distinct mature T-cell lineage with the capacity to proliferate in response to receptor-mediated signals, and to display non-major histocompatibility complex (MHC)-restricted cytolysis. Critical to understanding the function of this T-cell subset is the identification of the ligand(s) recognized by TCR gamma delta. Here we describe an alloreactive CD3+CD4-CD8-TCR gamma delta-expressing, TCR alpha beta-negative, T-cell line that manifests MHC-linked recognition specificity for both proliferation and cytotoxicity. Our results suggest that T cells expressing TCR gamma delta are capable of self-non-self MHC discrimination and that they can undergo MHC-influenced selection during differentiation like TCR alpha beta-expressing T cells.     

Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation

During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.     

Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes

The intrathymic differentiation process by which precursor cells derived from the bone marrow develop into immuno-competent T lymphocytes is poorly understood. Most thymocytes express both CD4 and CD8 accessory molecules, yet little is known about either the function of these molecules or the responsiveness of the CD4+8+ double positive thymocytes that bear them. Here, we address the possibility that CD4 engagement influences T-cell receptor (TCR) expression on developing thymocytes. We engaged CD4 molecules on murine thymocytes by in vivo injection of an anti-CD4 monoclonal antibody, which reduced the surface expression of CD4 on CD4+ thymocytes. More importantly, CD4 engagement also affected TCR expression on CD4+ thymocytes, but the effect on CD4+8+ double positive and CD4+8- single positive thymocytes was very different. CD4+8+ thymocytes responded to CD4 engagement by dramatically increasing surface expression of TCR, whereas CD4+8- thymocytes decreased surface expression of TCR. These results demonstrate that the effect of CD4 engagement on TCR expression is dependent upon the developmental state of the responding thymocyte, and, most interestingly, results in increased TCR expression by double positive thymocytes.     

Deletion of self-reactive thymocytes occurs at a CD4+8+ precursor stage

As T cells develop in the thymus, they become tolerant of self-antigens. A major advance in the understanding of how this process occurs was the direct demonstration that cells bearing autoreactive T-cell receptors (TCRs) are physically eliminated from the population of functionally mature T cells present in both the thymus and periphery. We have sought to determine the developmental stage at which autoreactive T cells are eliminated by examining the expression of V beta 17a anti-I-E TCRs under various experimental conditions. In vivo antibody blockage of the CD4 molecule on developing thymocytes of I-E+ C57BR mice was found to inhibit the deletion of V beta 17a-bearing cells from the CD4-8+ single positive thymocyte subset. This result provides strong evidence that deletion of potentially autoreactive T cells occurs at a CD4+8+ precursor stage, that the non-clonally distributed accessory molecules (CD4, CD8) are significant participants in the self-recognition process that leads to clonal elimination, and that thymic class II major histocompatibility complex (MHC) molecules can influence the repertoire of CD4-8+ cells.     

The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes

T lymphocytes recognize foreign molecules using the T-cell receptor (TCR), a disulphide-linked heterodimer closely associated with the CD3 polypeptide complex on the cell surface. The TCR alpha beta heterodimers seem largely responsible for the recognition properties of both helper (TH) and cytotoxic (TC) T cells. Recently, a second CD3-associated T-cell receptor heterodimer, gamma delta, has been described. Cells bearing the gamma delta receptor appear before those bearing alpha beta during thymic ontogeny and persist as a minor component (1-10%) of mature peripheral T cells. Their function is unknown. As there are a limited number of functional TCR V gamma gene segments, the size and potential diversity of the V delta repertoire is important for the number of different antigens that may be recognized by gamma delta heterodimers. The delta-chain locus is located 75 kilobases (kb) 5' to the TCR C alpha coding region, raising the possibility that the alpha and delta V-region repertoires may overlap. Also, analysis of rearrangements at the delta-chain locus in developing thymocytes shows distinct fetal and adult patterns indicating that there may be differences between the fetal and adult V delta repertoires. To address these questions, we have characterized a large number of delta-containing complementary DNA clones from adult double-negative thymocytes (CD4-8-), an immature population that is enriched for gamma delta-bearing cells. We find that a limited number of V delta sequences are used, showing little overlap with known adult V alpha s and differing significantly from fetal V delta s. But as two D elements may participate simultaneously in V delta gene assembly, and random nucleotides may be added at any one of three junctional points, the potential number of different delta chains that can be made in the adult thymus is very large (approximately 10(13)).     

A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.     

Peptide sequences of T-cell receptor delta and gamma chains are identical to predicted X and gamma proteins

Although most mature peripheral T lymphocytes express a major histocompatibility complex restricted, CD3-associated, antigen receptor (TCR) which has been well characterized, some T cells carry a different CD3-associated heterodimer on their surface. One of the two disulphide-linked chains of this putative second receptor, which in mice has relative molecular mass (Mr) 35,000 (35K), has been identified as a product of the group of gamma genes. The other chain, termed delta (Mr 45K in mice), is not as well characterized. Although gamma/delta-bearing cells are a minor subset among peripheral T lymphocytes, they are the only CD3+ cells in the thymus early in ontogeny. Taking advantage of these kinetics, we have generated gamma/delta-bearing hybridomas, using a new TCR alpha chain-negative variant of the AKR thymoma BW5147 as tumour parent, fetal thymocytes as normal cell partners, and an anti-CD3 monoclonal antibody (mAb) as screening reagent. Gamma and delta chains from one of these hybrids have been purified and partially sequenced. The sequences obtained indicate that delta is indeed identical to the polypeptide encoded by the recently described gene X, as suggested by Chien et al.