The apoptotic protease-activating factor 1-mediated pathway of apoptosis is dispensable for negative selection of thymocytes

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1(-/-) mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1(-/-) thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1(-/-) thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1(+/-) thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1(-/-) thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.     

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.     

Elevated Mcl-1 inhibits thymocyte apoptosis and alters thymic selection

T cells developing in the thymus undergo rigorous positive and negative selection to ensure that those exported to peripheral lymphoid organs bear T-cell receptors (TCRs) capable of reacting with foreign antigens but tolerant of self. At each checkpoint, whether a thymocyte survives or dies is determined by antiapoptotic and proapoptotic Bcl-2 family members. We used Mcl-1 transgenic (tg) mice to investigate the impact of elevated expression of antiapoptotic Mcl-1 on thymocyte apoptosis and selection, making a side-by-side comparison with thymocytes from BCL-2tg mice. Mcl-1 was as effective as Bcl-2 at protecting thymocytes against spontaneous cell death, diverse cytotoxic insults and TCR–CD3 stimulation-driven apoptosis. In three different TCR tg models, Mcl-1 markedly enhanced positive selection of thymocytes, as did Bcl-2. In H-Y TCR tg mice, elevated Mcl-1 and Bcl-2 were equally effective at inhibiting deletion of autoreactive thymocytes. However, in the OT-1tg model where deletion is mediated by a peripheral antigen whose expression is regulated by Aire, Mcl-1 was less effective than Bcl-2. Thus, the capacity of Mcl-1 overexpression to inhibit apoptosis triggered by TCR stimulation apparently depends on the thymocyte subset subject to deletion, presumably due to differences in the profiles of proapoptotic Bcl-2 family members mediating the deletion.     

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

The critical role of LIGHT, a TNF family member, in T cell development.

Negative selection refers to the selective deletion of autoreactive thymocytes but its molecular events have not been well defined. In this study, we demonstrate that a cellular ligand for herpes virus entry mediator and lymphotoxin receptor (LIGHT), a newly identified member of the TNF superfamily, may play a critical role in negative selection. Using TCR transgenic mice, we find that the blockade of LIGHT signaling in vitro and in vivo prevents negative selection induced by peptide and intrathymically expressed Ags, resulting in the rescue of thymocytes from apoptosis. Furthermore, the thymi of LIGHT transgenic mice show severe atrophy with remarkably reduced CD4(+)CD8(+) double-positive cells caused by increased apoptosis, suggesting that LIGHT can delete immature T cells in vivo. Taken together, these results demonstrate a critical role of LIGHT in thymic negative selection of the T cell repertoire.     

Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions

Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection.     

The affinity/avidity and length of exposure to the deleting ligand determine dependence on CD28 for the efficient deletion of self-specific CD4+CD8+ thymocytes

Whether the CD28/B7 signaling pathway is essential for the negative selection of immature CD4+CD8+ (DP) thymocytes expressing self-specific alphabeta TCRs is a controversial issue. In this study we examined the role of CD28 in the deletion of thymocytes that express either the H-Y or the 2C transgenic TCR. In H-2(b) male mice that expressed the H-Y TCR, negative selection of DP H-Y TCR+ thymocytes occurred very efficiently and this deletion was unaffected by the CD28(-/-) mutation. In H-2(b) 2C mice, where the deletion of DP 2C TCR+ thymocytes occurred less efficiently, the CD28(-/-) mutation led to a higher recovery of DP thymocytes. Using an in vitro deletion assay, a requirement for the CD28 signaling pathway in the deletion of DP H-Y TCR+ thymocytes was evident at low, but not high, densities of the antigenic ligand. Similar results were also observed in an in vivo assay for the deletion of these thymocytes. Intraperitoneal administration of an anti-CD3epsilon mAb led to the intrathymic deletion of DP H-Y TCR+ thymocytes in a CD28-dependent manner at the 24-h time point. However, the CD28 dependence was less evident at the 40-h time point. These results indicate that the dependence on CD28 for the efficient deletion of self-specific thymocytes is determined by the concentration, affinity/avidity, and length of exposure to the deleting ligand.     

Fine tuning of TCR signaling by CD5

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.     

Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3epsilon Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3epsilon-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.     

Quantitative and qualitative adjustment of thymic T cell production by clonal expansion of premigrant thymocytes

In normal mice, single-positive thymocytes proliferate before being exported into the peripheral T cell pool. We measured the in vivo proliferation rates of mature thymocytes in several TCR transgenic mice. Different monoclonal TCR transgenic single-positive thymocytes proliferated at different rates in a given MHC context. Conversely, mature thymocytes expressing a given TCR, generated in mice of different MHC haplotypes, also showed different rates of proliferation. In p59(fyn)-deficient mice, the proliferation rate of mature thymocytes was diminished. Thus, premigrant thymocyte expansion is TCR mediated and depends on TCR affinity for self peptide/MHC ligands. In addition, we show that mature thymocyte expansion is clonotypic, increases the daily thymic T cell output, and modifies the TCR repertoire of newly produced T cells.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

The Functional Role of B7 Molecules on the Induction of Thymocyte Activation and Apoptosis

To evaluate the role of B7 on thymocyte activation and apoptosis, we took advantage of TCR transgenic mice in which the majority of thymocytes express a uniform TCR that is specific for ovalbumin. We also prepared Chinese hamster ovary (CHO) cells expressing B7 and appropriate class II molecules. We found that the apoptosis of double-positive thymocytes by TCR-mediated signaling, which presumably represents negative selection, requires a costimulatory signal provided by B7-1 or B7-2. The requirement of B7-1 costimulation for the apoptosis of thymocytes does not change in either low or high antigenic peptide loading. We also demonstrated that two signals through TCR and CD28 augmented the proliferation of thymocytes, and the requirement of CD28-mediated signal by B7-1 or B7-2 for thymocyte proliferation became less evident when high doses of antigenic peptide were loaded, indicating that the intensity of TCR-mediated signal determines the requirement of B7-mediated second signal for thymocyte proliferation.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

A CD8/Lck transgene is able to drive thymocyte differentiation

Efficient development of thymocytes requires participation of a CD8 or CD4 coreceptor in the TCR:MHC interaction. Both CD8 and CD4 coreceptor cytoplasmic domains associate with Lck. In this study, we attempted to delineate the role of CD8alpha-associated Lck in driving CD8 single positive (SP) thymocyte development. We used a chimeric molecule encoding the extracellular and transmembrane domains of CD8alpha fused to full-length Lck. In mice deficient for CD8alpha and transgenic for 2C, a MHC class I-restricted TCR, robust reconstitution of CD8 SP thymocytes occurred both centrally and peripherally. The reconstituted CD8 SP population was phenotypically and functionally comparable to 2C wild-type counterparts expressing endogenous CD8alpha. A CD8alpha/Lck kinase-dead chimera also resulted in reconstitution of CD8 SP thymocytes. Our results suggest that CD8alpha-associated Lck is sufficient to drive CD8 SP thymocyte development. Furthermore, this CD8 SP development may not necessarily depend on Lck kinase activity.