DEC-205 as a marker of dendritic cells with regulatory effects on CD8 T cell responses

We have previously reported that a population of lymphoid-related CD8alpha(+) DEC-205(+) dendritic cells (DC) from mouse spleen have 'regulatory' effects on the T cells they activate. CD8 T cells produce IL-2 and give a sustained proliferative response to allogeneic CD8alpha(-) DEC-205(-) splenic DC, but produce little IL-2 and give a limited response to allogeneic CD8(+) DEC-205(+) splenic DC. Although CD8alpha and DEC-205 correlate closely among splenic DC, lymph nodes (LN) include a large population of CD8alpha(low) DEC-205(high) DC. By i.v. transfer of purified thymic early lymphoid precursors into irradiated recipient mice we now demonstrate that these CD8alpha(low) but DEC-205(high) LN DC can be the progeny of a lymphoid precursor population, apparently corresponding to the CD8alpha(high) DEC-205(high) DC progeny of the same precursors in spleen and thymus. By culture of the separated, purified DC with allogeneic CD8 T cells we demonstrate that the CD8alpha(low) DEC-205(high) DC of LN are also functionally equivalent to the CD8alpha(high) DEC-205(high) DC of spleen. Therefore, DEC-205 but not CD8alpha serves to segregate functionally distinct DC types in LN. However, DC isolated from the spleens of genetically manipulated DEC-205(null) mice and separated on the basis of CD8alpha expression have a similar capacity to stimulate CD8 T cells as their heterozygous littermate controls, with the CD8alpha(+) but now DEC-205(null) DC still giving restricted responses. In conclusion, high expression of DEC-205 appears to be a good marker of the lymphoid-related regulatory type of DC, but DEC-205 itself is not responsible for transmitting negative signals to the T cells.     

CD8- dendritic cells preferentially cross-present Saccharomyces cerevisiae antigens

Mouse splenic dendritic cell (DC) subsets possess distinct antigen-presentation abilities. CD8(+) DC are specialized in cross-presentation of antigens to CD8(+) T cells, whereas CD8(-) DC are more efficient in antigen presentation to CD4(+) T cells. In this study, we examined the capacity of CD8(+) and CD8(-) DC subsets to present fungal antigens in MHC class I and II molecules to CD8(+) and CD4(+) T cells, respectively. We used ovalbumin-expressing Saccharomyces cerevisiae (yeast-OVA) as a fungal model system. Both CD8(+) and CD8(-) DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin-1. In addition, both DC subsets induced similar OVA-specific CD4(+) T cell proliferation after incubation with yeast-OVA. However, the induction of OVA-specific CD8(+) T cell activation was largely restricted to the CD8(-) DC subset. Furthermore, only CD8(-) DC produced cytokines such as IL-10 and TNF-alpha and increased IL-23p19 and IL-23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo.     

The proliferative response of CD4 T cells to steady-state CD8+ dendritic cells is restricted by post-activation death

CD8(+) splenic dendritic cells (DCs) from steady-state mice are less effective than the CD8(-) DC subset in their capacity to stimulate CD4 T cell proliferation in culture. However, we found that the two DC subtypes were equally potent at activating CD4 T cells, based on up-regulation of CD69 and CD25 expression. Also, we found no difference in the rate of T cell death prior to entry into the first division. We then tracked carboxyfluorescein diacetate succinimidyl ester-labeled T cells and employed a quantitative model to assess in detail the CD4 T cell expansion process in response to stimulation with CD8(+) or with CD8(-) DCs. The time required for most T cells to replicate their DNA prior to the first division was similar in both DC cultures. However, progression of the CD4 T cell population through subsequent divisions was reduced in CD8(+) DCs compared with CD8(-) DC culture. This was associated with an increased loss of viable T cells at each division. Post-activation, division-associated T cell death is therefore a major factor in the reduced response of CD4 T cells to CD8(+) DCs.     

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.     

Differential capacity of CD8α+ or CD8α− dendritic cell subsets to prime for eosinophilic airway inflammation in the T-helper type 2-prone milieu of the lung

BACKGROUND: Different subsets of dendritic cells (DCs), identified in mouse spleen by their differential expression of CD8 alpha, can induce different T-helper (Th) responses after systemic administration. CD8 alpha(-) DCs have been shown to preferentially induce Th type 2 (Th2) responses whereas CD8 alpha(+) DCs induce Th1 responses. OBJECTIVE: To study if these DC subsets can still induce different Th responses in the Th2-prone milieu of the lung and differentially prime for eosinophilic airway inflammation, typical of asthma. METHODS: Donor mice first received daily Flt3L injections to expand DC numbers. Purified CD8 alpha(+) or CD8 alpha(-) splenic DCs were pulsed with ovalbumin (OVA) or phosphate-buffered saline and injected intratracheally into recipient mice in which carboxyfluorescein diacetate succinimidyl ester-labelled OVA-specific T cell receptor transgenic T cells had been injected intravenously 2 days earlier. T cell proliferation and cytokine production of Ag-specific T cells were evaluated in the mediastinal lymph nodes (MLNs) 4 days later. The capacity of both subsets of DCs, to prime for eosinophilic airway inflammation was determined by challenging the mice with OVA aerosol 10 days later. RESULTS: CD8 alpha(-) DCs migrated to the MLN and induced a vigorous proliferative T cell response accompanied by high-level production of IL-4, IL-5, IL-10 and also IFN-gamma during the primary response and during challenge with aerosol, leading to eosinophilic airway inflammation. In the absence of migration to the MLN, CD8 alpha(+) DCs still induced a proliferative response with identical levels of IFN-gamma but reduced Th2 cytokines compared with CD8 alpha(-) DCs, which led to weak eosinophilic airway inflammation upon OVA aerosol challenge. Unpulsed DCs did not induce proliferation or cytokine production in Ag-specific T cells. CONCLUSION: CD8 alpha(-) DCs are superior compared with CD8 alpha(+) DCs in inducing Th2 responses and eosinophilic airway inflammation in the Th2-prone environment of the lung.     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

IL-2 down-regulates the expression of TCR and TCR-associated surface molecules on CD8(+) T cells.

CD8(+) T cells are known to down-regulate the TCR complex upon ligation with its cognate MHC class I-peptide complex. In the present report, we demonstrate that stimulation of CD8(+) T cells with cytokines also leads to down-regulation of the TCR complex and TCR-associated surface molecules. A significant reduction of TCRalpha beta, CD3, CD8alpha and CD8beta surface expression was observed when CD8(+) T cells were cultured in IL-2 and to a lesser extent in IL-4 or IL-15. The down-regulation was apparent after 2 days of culture and was observed at IL-2 concentrations as low as 10 U/ml. Using TCR transgenic mice, we found that the down-regulation was associated with a decreased affinity of CD8(+) T cells to MHC class I-peptide complexes, as determined by MHC class I tetramer staining. Furthermore, the antigen-specific proliferation of IL-2-pre-activated CD8(+) T cells was significantly reduced compared to naive CD8(+) T cells or to CD8(+) T cells previously stimulated with peptide-pulsed dendritic cells. Moreover, only CD8alpha(high) but not CD8alpha(low) cells sorted from IL-2-activated CD8(+) T cells proliferated in response to specific antigen, although both subsets proliferated equally well to IL-2. Taken together, these data suggest that the down-regulation of TCR components and a subsequent decrease in affinity towards MHC class I-peptide complexes may be a mechanism by which TCR-dependent proliferation of non-specifically activated CD8(+) T cells is avoided.     

IL-4 influences the differentiation and the susceptibility to activation-induced cell death of human naive CD8+ T cells

It is now well established that the cytokine environment influences the activation, differentiation, proliferation and death of T lymphocytes during the primary response to antigen. Using an in vitro model, we investigated the influence of IL-4, added at the onset of TCR stimulation, on phenotypic and functional markers of naive CD8+ T cell activation including the up-regulation of activation markers, proliferation as well as the susceptibility to activation-induced cell death (AICD). We report that IL-4, unlike IL-2 added at the onset of repeated TCR stimulation of naive CD8+ T cells prevents AICD, in part due to its ability to maintain the level of the survival-related protein Bcl-2. Moreover, TCR-triggered activation of naive CD8+ T cells in the presence of IL-4 leads to the development of a CD8+ T cell subset that proliferates normally, but which fails to exhibit characteristic activation parameters such as the up-regulation of CD25 and Granzyme B. Taken together, these results demonstrate that exposure to IL-4 during primary activation influences CD8+ T cell differentiation by inducing the development of a sub-population of AICD-resistant, proliferation-competent cells that do not show some of the typical features of CD8+ T cell activation.     

CD8alpha dendritic cells and immune protection from experimental allergic encephalomyelitis

Dendritic cells (DC) represent a phenotypically heterogeneous population endowed with two important biological functions, immunity and tolerance. Here we report that the injection of splenic CD8alpha(+) DC, derived from rats with experimental allergic encephalomyelitis (EAE), delayed the onset and suppressed the severity of EAE in Lewis rats. This was accompanied by the lack of magnetic resonance imaging (MRI) lesions in the brain and spinal cord and by reduced numbers of inflammatory cells within the central nervous system. Injection of CD8(alpha+) DC inhibited T cell proliferation that may relate to increased interferon (IFN)-gamma and nitric oxide production. Although CD8(+)CD28(-) suppressor T cells, apoptotic cells and co-stimulatory molecules were not altered, CD4(+) T cells expressing interleukin (IL)-10 were augmented in rats receiving CD8alpha(+) DC compared to rats receiving total DC or medium. These results demonstrate that rat splenic CD8alpha(+) DC could provide a cellular basis for a novel, individualized immunotherapy using autologous DC as a complement to conventional therapy in diseases with an autoimmune background such as multiple sclerosis.     

Reduced antigen concentration and costimulatory blockade increase IFN-gamma secretion in naive CD8+ T cells

CD8+ T cells are killer cells but also major producers of IFN-gamma. We have investigated the effects of peptide antigen titration and costimulatory blockade on IFN-gamma production and proliferation by naive CD8+ T cells. Mature dendritic cells (DC) pulsed with high amounts of agonist peptide triggered proliferation but little IFN-gamma secretion in individual T cells. In contrast, immature DC pulsed with similar amounts of peptide induced IFN-gamma secretion in a larger fraction of T cells but triggered less proliferation. Blocking B7.2 or lowering the amount of peptide on mature DC led to a response similar to that induced by immature DC, suggesting that differences in stimulatory strength were responsible for the different responses. Using splenic antigen-presenting cells (APC) we demonstrate that reducing the amount of peptide in combination with B7 blockage enhanced IFN-gamma secretion and decreased proliferation in naive CD8+ T cells in an additive way. Our data suggest that IFN-gamma secretion and proliferation are independently and inversely controlled by stimulatory strength in naive CD8+ T cells. This may enable CD8+ T cells to respond with IFN-gamma secretion to immature APC with few peptide ligands consistent with an early immunoregulatory role of CD8+ T cells.     

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.     

CD8 alpha is an activation marker for a subset of peripheral CD4 T cells

Rat CD4 T lymphocytes express CD8 alpha upon activation. Here, we show that double-positive cells express CD8 alpha alpha homodimers, and we study their phenotype and function. Most activated CD4(+) lymphocytes expressing CD8 alpha are recent thymic emigrants. Accordingly, most activated CD4 single-positive thymocytes express CD8 alpha, and thymectomy and aging decrease the frequency of CD4(+)CD8 alpha(+) lymphocytes. However, CD8 induction is not restricted to CD4(+) recent thymic emigrants. CD4(+)CD8 alpha(+) and CD4(+)CD8 alpha(-)cells were generated in vitro from naive or from primed donors and, to study their function, were transferred to normal rats. Both cell types helped primary humoral responses, but only CD4(+)CD8 alpha(-) cells promoted secondary responses. Thus, memory CD4 T cells mediating antibody responses and some naive CD4(+) lymphocytes do not express CD8 alpha. In addition, CD4(+)CD8 alpha(+) cells produce mainly Th1 cytokines while CD4(+)CD8 alpha(-) cells produce IL-10 and showed a sustained proliferative response. Hence, CD8 alpha expression after activation distinguishes two distinct CD4 T cell subsets.     

Functional expression of indoleamine 2,3-dioxygenase by murine CD8 alpha(+) dendritic cells.

Immunoregulatory antigen-presenting cells (APC) play an important role in maintaining T cell homeostasis and self-tolerance. In particular, recent evidence demonstrates a role for inhibition of T cell proliferation by macrophage tryptophan catabolism involving the activity of the enzyme indoleamine 2,3-dioxygenase (IDO). Dendritic cells (DC) have also been shown to exert immunoregulatory effects mediated by tryptophan catabolism and to cause T cell apoptosis. In the present study, we have comparatively analyzed the expression of IDO activity by murine macrophages and splenic DC. By means of PCR, Western blotting and measurements of enzyme functional activity, we obtained evidence that, different from macrophages, DC constitutively express IDO. Following activation by IFN-gamma, the latter cells, in particular the CD8 alpha(+) subset, exhibit high functional activity and, unlike macrophages, mediate apoptosis of T(h) cells in vitro. Therefore, in the mouse, CD8 alpha(+) DC may be unique APC capable of fully expressing the IDO mechanism functionally.     

CTL priming by CD8(+) and CD8(-) dendritic cells in vivo

Two distinct developmental pathways are driving the formation of myeloid- and lymphoid-related dendritic cells (DC) which differ in anatomical localization and phenotype. In terms of function, it has been hypothesized that only the myeloid-related CD8(-) DC are able to initiate immune responses, whereas the lymphoid-related CD8(+) DC have been suggested to induce tolerance. Here we show that both subsets activate CD8(+) T cells in vitro and induce protective anti-viral CTL responses in vivo. Thus, vaccine strategies using peptide-pulsed DC do not have to take into account DC subsets for priming.     

Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.     

IFN-alpha-conditioned dendritic cells are highly efficient in inducing cross-priming CD8(+) T cells against exogenous viral antigens

Dendritic cells (DC) generated after a short-term exposure of monocytes to IFN-alpha and GM-CSF (IFN-DC) are highly effective in inducing cross-priming of CD8(+ )T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN-DC and IL-4-derived DC. Both DC types efficiently cross-presented soluble HCV NS3 protein to the specific CD8(+) T cell clone, even though IFN-DC were superior in cross-presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV-1 and injected into hu-PBL-SCID mice, the generation of virus-specific CD8(+ )T cells was markedly higher in animals immunized with IFN-DC than in mice immunized with CD40L-matured IL-4-DC. Of interest, in experiments with purified CD8(+ )T cells, IFN-DC were superior with respect to CD40L-matured IL-4-DC in inducing in vitro cross-priming of HIV-specific CD8(+ )T cells. This property correlated with enhanced potential to express the specific subunits of the IL-23 and IL-27 cytokines. These results suggest that IFN-DC are directly licensed for an efficient CD8(+) T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL-12 family cytokines.     

Role of CD8alpha+ and CD8alpha- dendritic cells in the induction of primary immune responses in vivo.

Data from adoptive transfer of mature dendritic cells (DC) indicate that they are responsible for the induction of primary immunity. Two subclasses of DC have been recently identified in spleen that differ in their phenotype and in certain regulatory features. In vitro, both subsets have the capacity to activate naive T cells, although CD8a+ DC have been shown to induce T cell apoptosis and to stimulate lower levels of cytokines compared with CD8alpha- DC. The objective of this study was to analyze the function of these distinct DC types in vivo. Our results show that both subsets, pulsed extracorporeally with antigen and injected in the footpads of syngeneic mice, sensitize an antigen-specific T cell primary response. However, CD8alpha+ cells trigger the development of Thl-type cells, whereas CD8alpha- DC induce a Th2-type response. These observations suggest that the Th1/Th2 balance in vivo is regulated by the antigen-presenting-cells of the primary immune responses.     

Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment

Memory T cells can be classified as central memory (T(CM), CD45RA(neg)CCR7(+)), effector memory (T(EM), CD45RA(neg)CCR7(neg)), and terminally differentiated cells (T(TD), CD45RA(+)CCR7(neg)) with different homing and effector capacities. In 101 healthy subjects aged from 5 to 96 years, distinct dynamics were evidenced between circulating CD4(+) and CD8(+) T cell populations. Naive CD4(+) and CD8(+) T cells decreased linearly with age, CD8(+) twice more rapidly. Memory cells outnumbered naive cells on average at 37.4 in the CD4(+) and 29.5 years of age in the CD8(+) pool. CD4(+) T(CM) and T(EM) cells were positively correlated and increased linearly at a similar rate with age, while CD4(+) T(TD) remained rare. CD8(+) T(EM) and T(TD) accumulated linearly with age, while T(CM) increased only slightly, and each memory subset was negatively correlated to the two others. Almost all CD8(+) T(TD) and some CD8(+) T(EM) had lost CD28 expression. Despite different dynamics, each individual CD4(+) naive and memory subset was correlated to the synonymous CD8(+) subset. Half of the subjects aged 65 years or older were characterized by extremely reduced CD8(+) naive and increased CD8(+) T(TD) cell counts, which could indicate an acceleration of the decay of the immune system from this age onward.     

Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.     

Human in vivo-activated CD45R0(+) CD4(+) T cells are susceptible to spontaneous apoptosis that can be inhibited by the chemokine CXCL12 and IL-2, -6, -7, and -15

The number of T cells that have undergone proliferation after antigen stimulation in vivo must be controlled to prevent excessive accumulation of T cells, autoimmunity, and T cell neoplasia. We describe here that primary human adenotonsillar memory phenotype CD45R0(+) CD4(+) T cells, but not adenotonsillar naive-phenotype CD45RA(+) CD4(+) T cells, or peripheral blood naive or memory CD4(+) T cells, express high levels of activation-associated antigens CD38, CD69, CD71, and HLA-DR. These in vivo-activated CD45R0(+) CD4(+) T cells were susceptible to spontaneous and rapid apoptosis in vitro. Apoptosis could not be inhibited by the disruption of Fas-Fas ligand engagement or by the pan-caspase inhibitor ZVAD. Cross-linking of the T cell antigen receptor did not rescue cells from apoptosis. Apoptosis could be partially inhibited by the chemokine CXCL12/SDF-1, by IL-6, and by the IL-2 receptor common gamma chain-signaling cytokines IL-2, -7, and -15. Inhibitors of phosphatidylinositol 3-kinase accelerated apoptosis. We conclude that after in vivo activation of CD45R0(+) CD4(+) T cells, the cells experience a period of intrinsically elevated sensitivity to apoptosis and that multiple external signals control their survival.