Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widelyused in transplantation medicine. A major effect of CsA is inhibitionof the differentiation of immature double-positive (DP) CD4⁺CD8⁺thymocytes into mature single-positive (SP) CD4⁺CD8^– orCD4^–CD8⁺thymocytes. The mechanisms underlying the changesin CD4/CD8 expression during normal differentiation of thymocytesand the way CsA interferes with this differentiation processare still unknown. Here we show that protein kinase C (PKC)activation by phorbol 12-myristate 13-acetate (PMA) causes adecrease of both CD4 and CD8 expression at the cell surfacelevel and at the mRNA level in a CD4⁺CD8⁺ T cell line and infreshly isolated thymocytes. A PKC inhibitor, staurosporin,interferes with the differentiation from DP to SP in fetal thymusorgan culture system. These data suggest that the alternationof CD4/CD8 expression from DP to SP is dependent on PKC activation.CsA blocks this decrease of CD4/CD8 expression by PMA in vitro.Moreover, this PMA effect is also blocked by treatment withcycloheximlde. These results suggest that the reduction of CD4/CD8expression requires de novo synthesis of a protein(s) inducedin response to a signal conveyed by activated PKC. CsA may blockthe transition from DP to SP by inhibition of CD4/CD8 down-regulationinduced by PKC activation.     

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.     

CD 69 expression during selection and maturation of CD4+8+ thymocytes

We have analyzed the inducibility of protein kinase C (PKC)-dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4⁺8⁺ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross-linking even after spontaneous in vitro up-regulation of their TCR level which resulted in enhanced Ca⁺⁺ flux. In contrast, a small proportion of CD4⁺8⁺TCR^low and most TCR^high (CD4⁺8⁺ and CD4⁺8⁺) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross-linking. Unlike the anti-TCR antibody, phorbol 12-myristate 13-acetate - a direct activator of PKC - induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4⁺8⁺ thymocytes results in coupling of TCR-mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4⁺8⁺ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR-mediated signals.     

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.     

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.     

Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2^−/− backgrounds were developmentally arrested at the CD4⁺CD8⁺ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.     

Irreversible Suppression of CD8 Expression in CD4-CD8+ Thymocytes Upon in vitro Stimulation

CD8 (Ly-2) expression was suppressed in purified murine CD4-CD8⁺ thymocytes at the mRNA level upon continuous stimulation with PMA and ionomycin in the presence of rIL-2. The level of CD8 expression on CD4^?CD8⁺ thymocytes was reduced gradually during the culture and a majority of them turned into CD4^? CD8^? cells after 48 hr. This suppression was not transient, since CD8 expression was not recovered on these cells in additional 48 hr of culture without PMA and ionomycin. The suppression was dependent on the concentrations of PMA and ionomycin, and inhibited by adding an immunosuppressant, CsA to the culture. Treatment with either PMA or ionomycin alone did not induce suppression of CD8. Crosslinking of CD3-? chains also induced suppression of CD8 for a part of CD4^?CD8⁺ thymocytes. Interestingly, CD8 expression was hardly suppressed in CD4^?CD8⁺ peripheral T lymphocytes, suggesting that the mechanisms of suppression of CD8 is developmentally regulated. We propose that the suppression of CD8 expression at CD4^?CD8⁺ stage involves an additional mechanism of negative selection of thymic T cells.     

Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

Positive selection of CD4+ T cells by TCR-specific antibodies requires low valency TCR cross-linking: implications for repertoire selection in the thymus

The developmental fate of immature CD4⁺ 8⁺ thymocytes is determined by intrathymic signals transduced by surface TCR complexes. In particular, TCR signals are required for immature CD4⁺ 8⁺ thymocytes to further differentiate into CD4⁺ 8⁻ or CD4⁻ 8⁺ T cells, a process referred to as positive selection. It is generally thought that positive selection results from low affinity TCR interactions with self antigens which engage the relatively few surface TCR complexes that are on immature CD4⁺ 8⁺ thymocytes. However, we now demonstrate with TCR-specific antibodies that positive selection of CD4⁺ T cells requires low valency cross-linking of surface TCR complexes on immature thymocytes. That is, positive selection signals are only generated within a narrow range of TCR cross-linking: cross-linking either too few or too many surface TCR complexes fails to signal positive selection. We interpret these results as indicating that positive selection of CD4⁺ T cells is not signaled by low affinity TCR interactions per se, but rather can be signaled by any combination of TCR affinity and ligand density that induces low valency TCR cross-linking on immature thymocytes.     

GATA-3 expression is controlled by TCR signals and regulates CD4/CD8 differentiation

GATA-3 is expressed at higher levels in CD4 than in CD8 SP thymocytes. Here we show that upregulation of GATA-3 expression in DP thymocytes is triggered by TCR stimulation, and the extent of upregulation correlates with the strength of the TCR signal. Overexpression of GATA-3 or a partial GATA-3 agonist during positive selection inhibits CD8 SP cell development but is not sufficient to divert class I-restricted T cell precursors to the CD4 lineage. Conversely, expression of the GATA-3 antagonist ROG or of a GATA-3 siRNA hairpin markedly enhances development of CD8 SP cells and reduces CD4 SP development. We propose that GATA-3 contributes to linking the TCR signal strength to the differentiation program of CD4 and CD8 thymocytes.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

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.     

Reduced CD4 and CD8 Expression in Human Thymuses Treated with Soluble CD4

Recent data suggest that accessory molecules like CD4 and CD8 act as co-receptors in intrathymic T-cell development. Soluble CD4 (sCD4) molecules offer a novel experimental approach to investigate the relevance of CD4 interaction with its putative intrathymic receptor for T-cell maturation. We attempted to inhibit binding of surface CD4 on thymocytes to its intrathymic receptor competitively by introduction of human sCD4 into human thymus tissue cultures. Our results demonstrate that sCD4, while not affecting peripheral T-cell responses as shown in control experiments, significantly affects intrathymic development of T lymphocytes. Immature CD4CD8 double positive (DP) thymocytes responded with reduced expression of both CD4 and CD8 molecules. This phenomenon could be followed up to the stage of single positive (SP) thymocytes: density of CD4 molecules on CD4 SP thymocytes and, even more interestingly, CD8 expression on CD8 SP cells, were reduced, indicating that the effect observed in immature DP thymocytes persists during their further development. Beyond that, analysis of T-cell receptor (TCR) expression in the low density CD4CD8 DP population revealed a slight decrease of alpha beta-TCR surface expression, suggesting a possible role of CD4 engagement in the generation of TCR in man. Since sCD4 is considered a therapeutical agent in HIV infections, these findings are not only of basic but also of clinical interest.     

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.     

The frequency of double-positive thymocytes expressing an alphabeta TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing alphabeta T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Calpha(0/0) and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Calpha(0/0) thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12-14 weeks following BM inoculation, the frequency of TCR Calpha(0/0) DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.     

In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.