Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD⁺8⁺ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.     

Alterations during positive selection in the thymus of nackt CD4-deficient mice

The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.     

Antigen and glucocorticoid hormone (GC) induce positive selection of DP thymocytes in a TcR transgenic mouse model

Thymocyte maturation in the thymus is controlled by stromal and humoral components. Among the humoral regulators locally produced glucocorticoids (GCs) seem to have a key role in the positive selection of thymocytes. Our previous studies have shown that the administration of GCs or the stimulation through the CD3 complex can induce apoptosis of double positive (DP) cells, but the combined presence of these stimuli induces positive selection. In this work our aim was to investigate the effects of antigen exposure and synthetic GC hormone (dexamethasone, DX) administration on the selection processes of DP cells in TcR transgenic mice. In our model, AND—pigeon cytochrome c (PCC)-specific I-E^k (MHC-II) restricted Vβ3, V11 TcR expressing transgenic mice were treated with PCC, with high or low dose DX, or with PCC and DX together, followed by the analysis of total thymocyte numbers, thymocyte composition, with regard to their CD69, Vβ3 and Annexin V expression. The administration of PCC and/or DX for 2 days resulted in a decreased DP cell number and a significantly increased CD4 SP cell ratio. However, in both cases the total thymocyte numbers decreased. CD69 expression increased on both DP and CD4 SP cells after PCC and/or DX treatments. We found that after DX or combined treatment, the percentage of Annexin V positive cells increased. The ratio of Vβ3 TcR bearing DP thymocytes showed no change after DX or PCC administrations alone, but it decreased significantly after combined treatment. MHC-II bound PCC peptides in the presence of GCs enhanced the maturation of Vβ3+ DP cells into CD4 SP stage, therefore, the Vβ3− cells remained mostly in the DP immature stage. These data indicate that both antigen and low dose GC alone are capable of inducing positive selection of DP cells, but together they gave a stronger effect in promoting positive selection. From these we conclude that GCs influence the maturation and selection processes of thymocytes.     

Effect of phorbol ester and calcium ionophore on human thymocytes

Positive selection of immature thymocytes is a developmental process in which TCR ligation with low avidity induces generation of mature T cells. In mouse thymocytes, CD4(+)8(+) double-positive (DP) cells which were treated with a proper combination of calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) have been reported to differentiate into CD4 single positive cells. However, in human thymocytes the effects of PMA and ionomycin have remained unclear. Here we report that DP cells that were treated with PMA and ionomycin up-regulated bcl-2 and down-regulated CD1 expression. However, CD3 expression remained low. This treatment induced prolonged CD4 down-regulation in DP cells which was an effect also seen in mature peripheral blood T cells. PMA/ionomycin-treated DP cells showed high cell proliferation and resistance to dexamethasone-induced apoptosis. These results indicate that PKC activation and calcium elevation may be part of the biochemical signals that induce positive selection of human DP cells and the system described in this paper may be a useful model to study the signals involved in the selection of human thymocytes.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.     

Cross-linking of the TCR CD3 complex with CD4, CD8 or LFA-1 induces an anti-apoptotic signal in thymocytes: the signal is canceled by FK506

The immunosuppressant FK506 did not block differentiation ofdouble-negative thymocytes into double-positive (DP) cells,but interfered with differentiation of DP cells into maturesingle-positive cells In a fetal thymus organ culture system,suggesting that FK506 inhibits positive selection. The drugalso reduced the number of DP cells recovered after the culture.As positive selection depends on the inhibition of thymocyteapoptosis at Its DP stage by signaling through the TCR-CD3 complexand some of the accessory molecules, including CD4, CDS andLFA-1, we studied the possibility that FK506 enhanced apoptosisby itself or canceled the inhibition of apoptosis. The resultsIndicated that FK506 was hardly toxic or hardly affected antl-CD3-inducedDNA fragmentation in isolated thymocytes In vitro. On the otherhand, upon cross-linking TCR-CD3 together with CD4, CD8 or LFA-1,FK506 markedly enhanced DNA fragmentation and cytolysis. Thedrug, however, hardly or only slightly enhanced these responsesupon cross-linking TCR-CD3 together with CD2, CD28, Thy-1 orH-2K^d. Cross-linking of TCR-CD3 together with CD4, CD8 or LFA-1markedly Inhibited glucocorticoid-induced death and the inhibitionwas canceled by FK506. Furthermore, cross-linking of TCR-CD3together with LFA-1 enhanced Bcl-2 expression In DP cells. Theseresults suggest that cross-linking of TCR-CD3 with CD4, CD8or LFA-1 potentially induces both an apoptosis-lnducing signaland an FK506-sensttive anti-apoptotic signal, and that the lattersignal may be related to an essential signal for positive selection.     

In vitro differentiation and commitment of CD4+ thymocytes to the CD4+ lineage without TCR engagement

Thymocyte positive selection is based on protection of immatureCD4/CD8 double-positive (DP) thymocytes from apoptosis and theirdifferentiation into CD4 or CD8 single-positive (SP) cells.Intracellular signals essential for positive selection appearto be induced through the TCR and some of the accessory moleculesincluding LFA-1, CD4 and CD8 upon Interaction with thymic stromalcells. The signals, however, still remain to be identified.Since physiological levels of glucocorticoids potentially induceor enhance thymocyte apoptosis even in vivo, the signals arelikely to inhibit the apoptotic effect of glucocorticoids. Wehave previously shown that proper cross-linking of TCR-CD3 withLFA-1, CD4 or CD8 inhibited glucocortlcold-lnduced thymocyteapoptosis in vitro, and that a proper combination of the calciumionophore, ionomycin and the protein kinase C (PKC) activator,phorbol 12-myrlstate 13-acetate (PMA), mimicked the inhibitoryeffect. Here we determined whether this combination of ionomycinand PMA induces differentiation of isolated DP thymocytes fromnormal and TCR transgenic mice. We found that pretreatment ofDP thymocytes with ionomycin and PMA followed by 1 day cultureof the cells without the reagents resulted in the differentiationof the cells into CD4 SP and CD4⁺ CD8^lo T cells that have mostlycommitted to the CD4 lineage. The changes in expression of otherdifferentiation markers were also in good accordance with thoseassociated with positive selection, except the final maturation.The results indicate that moderate and transient increases inintracellular Ca²⁺ level and PKC activity induce differentiationand commitment of DP thymocytes to the CD4 lineage, and suggestedthat the biochemical pathway leading to positive selection isbased on a similar mechanism.     

Development of autoreactive diabetogenic T cells in the thymus of NOD mice

Type 1 diabetes results from destruction of pancreatic beta cells by beta cell-specific autoreactive T cells in the nonobese diabetic (NOD) mouse. Defects in thymic negative selection are thought to result in failure to delete potential beta cell-reactive T cells, contributing to the development of autoimmune diabetes. We investigated this possibility by comparing the deletion profile of double-positive (DP) thymocytes in NOD mice with diabetes-resistant strains of mice after anti-CD3 Ab treatment to trigger the TCR-mediated signaling pathway. We found that immature NOD CD4+CD8+ DP thymocytes have a lower activation threshold than C57BL/6 and Balb/c thymocytes. This was confirmed by showing that NOD DP thymocytes have a higher level of ERK and JNK phosphorylation. The low activation threshold of immature thymocytes resulted in rapid deletion of strongly activated immature DP thymocytes by negative selection, whereas weakly activated immature thymocytes differentiated more efficiently into CD69+CD3high DP thymocytes by positive selection. SP thymocytes, particularly CD4-CD8+ T cells that were efficiently generated from activated DP thymocytes, could induce severe insulitis and diabetes in NOD.scid mice. We conclude that the development of autoreactive diabetogenic T cells results from inordinate positive selection due to the low activation threshold of DP thymocytes in NOD mice.     

Reactive oxygen species induced by the deletion of peroxiredoxin II (PrxII) increases the number of thymocytes resulting in the enlargement of PrxII-null thymus

In the thymus, CD4+ or CD8+ single-positive (SP) thymocytes develop and mature by positive and negative selection or undergo "death by neglect". CD4+ or CD8+ SP then circulate to other lymphoid tissues. We have investigated the role of reactive oxygen species (ROS) in thymocyte development using peroxiredoxin II (PrxII)-null mice. The level ofROS in PrxII-null thymocytes is higher than that in wild-type mice. Deletion of the PrxII gene leads to enlargement of the thymus in young (9 weeks) and old (64 weeks) mice. The increased number ofthymocytes in PrxII-null thymus is related to reduced hypodiploid cell formation. For mice on a normal diet, the ratio of SP to double-positive (DP) thymocytes in thymus of PrxII-null mice is lower than that in wild-type mice. After food restriction, which leads to increased ROS production, this ratio becomes much higher in PrxII-null thymus. The amount of apoptosis, induced by food restriction orby the injection of dexamethasone, is consistently lower in PrxII-null thymocytes than in wild-type thymocytes. In the presence of low serum concentrations, PrxII-deleted T cells proliferate more vigorously after stimulation with concanavalin A. Phytohemagglutinin- or OKT3-stimulated proliferation of human peripheral blood mononuclear cells is also higher in the presence of lower serum concentrations. Collectively, the results suggest for the first time that thymocyte maturations and proliferations are regulated by ROS levels induced by the deletion of PrxII gene in vivo.     

Double-positive thymocytes resistant to antigen-MHC-induced negative selection lack active caspase

Thymocytes bearing autoreactive TCR are eliminated from the organism by a process termed negative selection. The molecular basis of this deletion has been recently shown to be a consequence of TCR-triggered activation of a caspase by certain peptide-MHC ligands in the immature CD4+CD8+ double-positive (DP) thymocyte subpopulation. Of note, the numerically minor TCRhigh DP thymocyte subpopulation, unlike the major TCRlow DP subset, is resistant to negative selection. Despite exposure to cognate peptide, TCRhigh DP thymocytes mature into single-positive thymocytes and are exported into the periphery. Here we investigated the mechanism by which these thymocytes escape negative selection. Using a cytochemical assay in conjunction with a caspase-specific affinity ligand, we demonstrate that the resistance of the TCRhigh DP thymocytes to negative selection correlates with the disappearance of TCR-triggered caspase activity in these cells. Thus thymocytes which have presumably begun the positive selection process inactivate the thymic caspase pathway and are no longer susceptible to negative selection.      

CD45 can act as a negative regulator for the transition from early to late CD4+ CD8+ thymocytes

The differentiation process from CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) stage is accompanied by vigorous proliferation. The resulting DP cells contain a sizable proportion of large cycling cells, but most DP cells are small resting cells. To explore the molecular mechanisms which regulate cell proliferation of DP thymocytes prior to further development, we used TCR-transgenic (Tg) mice with non-selecting MHC (Tg-Neut), which contain almost exclusively DP thymocytes that are not subject to either positive or negative selection. In Tg-Neut, the thymus contained DP cells of relatively large size, which showed higher extracellular signal-regulated kinase activity and enhanced responsiveness to mitogen compared to small DP cells. This indicates that all the large DP cells in the thymus are not positively selected and that they possess proliferative potential. When Tg-Neut mice were backcrossed with CD45 knockout mice (CD454-/- Tg-Neut), the thymus showed an increase of large DP cells and cycling cells, but a decrease of apoptotic cells. Furthermore, Bcl-2 expression and Jun N-terminal kinase activity, which are associated with resistance to apoptosis, were enhanced. These observations suggest that thymocyte proliferation in the DP stage is suppressed by a CD45-related process with regulation of mitogen-activated protein kinase and Bcl-2 unless DP cells receive TCR-mediated signals.     

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus

Thymic commitment of human FOXP 3⁺ regulatory T cells begins at the double‐positive (DP ) CD 4⁺ CD 8⁺ stage. In the current study, we show that interleukin‐2 promotes the development of FOXP 3⁺ thymocytes and enhances their survival at the DP phase. IL ‐2 increases the frequency of FOXP 3⁺ cells and promotes the Treg phenotype after TCR ‐mediated positive selection at the most mature DP stage. However, it has no effect on FOXP 3⁺ cells at the earlier maturation steps before positive selection. DP FOXP 3⁺ thymocytes are highly susceptible to cell death but IL ‐2 promotes their survival. The anti‐apoptotic protein BCL ‐2 (B Cell Lymphoma 2) is also upregulated by IL ‐2 at the most mature DP stage. In addition to IL ‐2, we identify IL ‐15 to have a significant role in the upregulating FOXP 3 and survival of Tregs at the DP phase. IL ‐7 also increases the expression of BCL ‐2 in the DP FOXP 3⁺ thymocytes. Our results indicate that common gamma chain cytokines IL ‐2, IL ‐7 and IL ‐15 promote the development of regulatory T cells at the most mature DP stage after TCR ‐mediated positive selection through suppressing cell death.     

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.     

The Ras/MAPK cascade and the control of positive selection

Immature double positive (DP) thymocytes bearing a T cell receptor (TCR) that interacts with self‐major histocompatibility complex (MHC) molecules receive signals that induce either their differentiation (positive selection) or apoptosis (negative selection). Furthermore, those cells that are positively selected develop into two different lineages, CD4 or CD8, depending on whether their TCRs bind to MHC class II or I, respectively. Positive selection therefore involves rescue from the default fate (death), lineage commitment, and progression to the single positive (SP) stage. These are probably temporally distinct events that may require both unique and overlapping signals. Work in the past several years has started to unravel the signaling networks that control these processes. One of the first pathways identified as important for positive selection was Ras and its downstream effector, the Erk mitogen‐activated protein kinase (MAPK) cascade. In this review we examine the factors that connect the TCR to the Ras/Erk cascade in DP thymocytes, as well as what we know about the downstream effectors of the Ras/Erk cascade important for positive selection. We also consider the possible role of this cascade in CD4/CD8 lineage development, and the possible interactions of the Ras/Erk cascade with Notch during these cell fate determination processes.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

Characterization of constitutive and strain-dependent subsets of CD45RA+ cells in the thymus.

We have previously shown that the occurrence of CD45RA+ adult mouse thymocytes is strain-dependent, e.g. constituting approximately 0.6% in C57BL/Icrf and approximately 2.5% in BALB/c (Huby, R. and Goff, L., 1992. Eur. J. Immunol. 22:1659). Here we show that irrespective of strain, the thymus contains approximately 0.6% CD45RA+ cells which are composed of slg+ B cells (approximately 0.4%), slg- CD4-CD8- cells (< 0.2%), and CD4+ CD8+ cells (< 0.2%). In some strains an additional CD45RA+ population, representing up to approximately 2% of all thymocytes, is present and has a CD4-CD8+ phenotype. It is this CD4-CD8+CD45RA+ subset which is responsible for the observed strain difference. In BALB/c mice, this additional population comprises approximately 90% of the CD45RA+ thymic cells. They are larger than the majority of thymocytes, with a size typical of mature, single positive cells (CD4+CD8- or CD4-CD8+). Further phenotyping for co-expression of other maturation markers showed them to be distinctive; they are CD3int-hi, i.e. as bright as other CD8 single positives, which are dimmer than CD4 single positives. In addition they are CD44hi, MEL-14dim and hi, Thy-1lo, HSAlo/-, and PNAlo, suggesting them to be amongst the most mature cells in the thymus. This was corroborated by their phenotypic similarity to CD45RA+ lymph node T cells. Furthermore, in BALB/c adult thymus sections, CD45RA+ cells are localized mainly in the medulla, consistent with a mature phenotype. Comparable with most mature thymocytes, cell cycle analysis revealed this subset to be composed of resting (G0/G1) cells. The CD4-CD8+CD45RA+ cells are amongst the most mature thymocytes and yet are indistinguishable from peripheral T cell counterparts; the possibilities that they are mature thymocytes due to exit the thymus, or that they may represent recirculating peripheral T cells, are discussed.     

A new function for LAT and CD8 during CD8‐mediated apoptosis that is independent of TCR signal transduction

The majority (>95%) of thymocytes undergo apoptosis during selection in the thymus. Several mechanisms have been proposed to explain how apoptosis of thymocytes that are not positively selected occurs; however, it is unknown whether thymocytes die purely by “neglect” or whether signaling through a cell‐surface receptor initiates an apoptotic pathway. We have previously demonstrated that on double positive thymocytes the ligation of CD8 in the absence of TCR engagement results in apoptosis and have postulated this is a mechanism to remove thymocytes that have failed positive selection. On mature single positive T cells CD8 acts as a co‐receptor to augment signaling through the TCR that is dependent on the phosphorylation of the adaptor protein, linker for activation of T cells (LAT). Here, we show that during CD8‐mediated apoptosis of double positive thymocytes there is an increase in the association of CD8 with LAT and an increase in LAT tyrosine phosphorylation. Decreasing LAT expression and mutation of tyrosine residues of LAT reduced apoptosis upon crosslinking of CD8. Our results identify novel functions for both CD8 and LAT that are independent of TCR signal transduction and suggest a mechanism for signal transduction leading to apoptosis upon CD8 crosslinking.