Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response.

CD8 T cells undergo autocrine IL-2-dependent proliferation upon TCR engagement and costimulation, but within 3-4 days, they become activation-induced nonresponsive (AINR) and display a split anergy. They can lyse targets and secrete IFN-gamma but they cannot produce IL-2 in response to TCR ligation and costimulation, due at least in part to an inability to up-regulate mitogen-activated protein kinases and IL-2 mRNA. Exogenous IL-2 can drive continued proliferation of AINR cells and nonresponsiveness is reversed within 1-2 days so that Ag-driven proliferation can resume. Mitogen-activated protein kinases and IL-2 mRNA can again be up-regulated, but "rewiring" has occurred so that these events no longer depend upon costimulation; TCR engagement is sufficient. Development of AINR appears to be a normal part of the differentiation program of CD8 T cells, providing a regulatory checkpoint to convert the initial helper-independent response to one that depends upon CD4 T cell help for continued expansion of the effector CTL. Once permission is given, in the form of IL-2, to pass this checkpoint, the CTL can make a prolonged response to persisting Ag in the absence of further CD4 T cell help.     

Signaling alterations in activation-induced nonresponsive CD8 T cells

Costimulation-dependent production and autocrine use of IL-2 by activated CD8 T cells results in initial clonal expansion, but this is transient. The cells quickly become anergic, unable to produce IL-2 in response to Ag and costimulation, irrespective of the form of costimulation. This activation-induced non-responsiveness (AINR) differs from "classical" anergy in that it results despite the cells receiving both signal 1 and signal 2. AINR cells can still proliferate in response to exogenous IL-2, but can no longer produce it. Other TCR-mediated events including cytolytic function and IFN-gamma production are not affected in the AINR state. To characterize the mechanism(s) responsible for lack of IL-2 production in CD8 T cells in the AINR state, microspheres bearing immobilized anti-TCR Abs or peptide-MHC complexes, B7-1, and ICAM-1 were used to provide well-defined stimuli to the cells. Comparison of normal and AINR cells revealed that in AINR cells extracellular signal-regulated kinase (ERK) is upregulated more transiently, Janus kinase activation is substantially reduced, and activation of p38 is eliminated. PMA and ionomycin restored proliferation and IL-2 production in AINR cells, indicating a signaling defect upstream of Ras and protein kinase C. Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2 mRNA expression and proliferation of both peptide-MHC/B7-1/ICAM-1-stimulated normal cells and PMA/ionomycin-stimulated AINR cells. Together these results demonstrate that activation of at least ERK and p38 is essential for IL-2 production by CD8 T cells and that up-regulation of these mitogen-activated protein kinases, along with Janus kinase, is defective in AINR cells.     

CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation.

CD8+ T cells stimulated in vitro with anti-TCR mAb and B7-1 or ICAM-1 produce IL-2 and clonally expand. Effector function is acquired within 3 days, but proliferation ceases and the cells begin to die by apoptosis. Stimulation in vivo with B7-1-expressing allogeneic tumor results in the same sequence of events with a comparable time course. In both cases, the cells become anergic within 3 or 4 days of responding; they can no longer respond by producing IL-2 and proliferating, but can still be stimulated to proliferate in response to exogenous IL-2. This activation-induced nonresponsiveness (AINR) is not simply a consequence of ongoing cell death; cytokines that promote survival (IL-7 or IFN-alpha) or proliferation (human IL-2) do not restore the ability to produce IL-2 in response to costimulation. Although similar to the anergy described for CD4+ T cell clones, AINR differs in that it results from an initial stimulation with both signal 1 and signal 2. AINR appears to be an aspect of the normal differentiation of fully stimulated CD8+ T cells. It is probably important in regulating CTL responses; it limits the initial T helper-independent response and converts it to a response that requires T cell help to be sustained and further expanded. When the initial helper-independent response is not sufficient to clear Ag, and if help is not available, AINR likely results in tolerance to the Ag.     

On CD28/CD40 ligand costimulation, common gamma-chain signals, and the alloimmune response

Activation and robust expansion of naive T cells often require T cell costimulatory signals and T cell growth factors. However, the precise growth and costimulation requirements for activation and expansion of CD4(+) and CD8(+) T cells in vivo in allograft response are still not clearly defined. In the present study, we critically examined the role of CD28/CD40 ligand (CD40L) costimulation and the common gamma-chain (gamma(c)) signals, a shared signaling component by receptors for all known T cell growth factors (i.e., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21), in activation and expansion of CD4(+) and CD8(+) T cells in the allogeneic hosts. We found that CD28/CD40L costimulation and the gamma(c) signals are differentially involved in proliferation and clonal expansion of CD4(+) and CD8(+) T cells in response to alloantigen stimulation. CD8(+) T cells are highly dependent on the gamma(c) signals for survival, expansion, and functional maturation, whereas in vivo expansion of alloreactive CD4(+) T cells is largely gamma(c) independent. T cell costimulation via CD28 and CD40L, however, is necessary and sufficient for activation and expansion of CD4(+) T cells in vivo. In a skin transplant model, blocking both CD28/CD40L and the gamma(c) pathways induced prolonged skin allograft survival. Our study provides critical insights that the CD4 and CD8 compartments are most likely governed by distinct mechanisms in vivo, and targeting both costimulatory and gamma(c) signals may be highly effective in certain cytopathic conditions involving activation of both CD4(+) and CD8(+) T cells.     

The roles of IL-12 in providing a third signal for clonal expansion of naive CD8 T cells

Stimulation of an effective in vitro or in vivo response by naive CD8 T cells requires three signals: TCR engagement, costimulation/IL-2, and a third signal that can be provided by IL-12. In addition to being required for acquisition of cytolytic function, IL-12 is required for optimal IL-2-dependent proliferation and clonal expansion. In experiments examining in vitro stimulation of naive CD8 T cells, IL-12 is shown to stimulate expression of the IL-2R alpha-chain (CD25) to much higher levels than are reached in response to just TCR and costimulation and/or IL-2. In addition, high CD25 expression is substantially prolonged in the presence of IL-12. As a consequence, the cells proliferate more effectively in response to low levels of IL-2. Examination of adoptively transferred TCR transgenic CD8 T cells responding to peptide Ag confirmed that IL-12 up-regulates CD25 in vivo, even when B7-mediated costimulation is largely blocked. TCR- and IL-2-dependent proliferation of CD8 T cells from mice deficient in CD25 was also found to increase in the presence of IL-12, indicating that CD25 up-regulation is not the only mechanism by which IL-12 increases clonal expansion of the cells. IL-2 and IL-12 both act to increase expression of both CD25 and the IL-12R, thus providing positive cross-regulation of receptor expression. These results suggest that when cross-priming dendritic cells present class I/Ag and costimulatory ligands, and produce IL-12, naive CD8 T cells will begin to produce IL-2 and both receptors will be optimally up-regulated to insure that an effective response is generated.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

Cutting edge: T cell requirement for CD28 costimulation is due to negative regulation of TCR signals by PTEN

Recent studies suggest that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays a critical role in the maintenance of self-tolerance. Using T cell-specific PTEN knockout mice (PTENDeltaT), we have identified a novel mechanism by which PTEN regulates T cell tolerance. We found that TCR stimulation alone, without CD28 costimulation, is sufficient to induce hyperactivation of the PI3K pathway, which leads to enhanced IL-2 production by naive PTENDeltaT T cells. Importantly, as a result of this increased response to TCR stimulation, PTENDeltaT CD4(+) T cells no longer require CD28 costimulation for in vitro or in vivo expansion. In fact, unlike wild-type T cells, PTENDeltaT CD4(+) T cells are not anergized by delivery of TCR stimulation alone. These data suggest that by negatively regulating TCR signals, PTEN imposes a requirement for CD28 costimulation, thus defining a novel mechanism for its role in self-tolerance.     

Cross-linking of 4-1BB up-regulates IL-13 expression in CD8(+) T lymphocytes

4-1BB, one of co-stimulatory molecules, is a member of TNF receptor superfamily and expressed on T cells upon TCR ligation. We have shown that 4-1BB is a co-stimulatory molecule enhancing cell cycle progression and inhibiting activation-induced cell death of CD8+ T cells by enhancing TCR signaling pathways. Here, we first report that the cross-linking of 4-1BB increased the expression of IL-13 mRNA and protein, and its secretion apparently via calcineurin, a Ca2+/calmodulin-dependent phosphatase. Ligation of 4-1BB with p815-m-4-1BBL evoked intracellular Ca2+ level in CD8+ T cells. CD8+ T cells express IL-13 receptor alpha1 mRNA. Incubation with anti-IL-13 blocking mAb reduced proliferation of CD8+ T cells enhanced by 4-1BB, and the treatment of CD3/4-1BB-ligated CD8+ T cells with recombinant IL-13 enhances cell proliferation, indicating that 4-1BB-induced IL-13 expression is partially responsible for the CD8+ T cell expansion in an autocrine or paracrine manner.     

Differential in vitro activation of CD8-CD4+ and CD4-CD8+ T lymphocytes by combinations of anti-CD2 and anti-CD3 antibodies

Purified peripheral blood T lymphocytes and the CD8-CD4+ and CD4-CD8+ T cell subsets, exhaustively depleted of APC have been studied for their capacity to respond to mAb directed against the CD3-Ti Ag-specific TCR complex and against the CD2 SRBCR. It is demonstrated that high affinity IL-2R can be readily induced by either anti-CD3 and/or anti-CD2 stimulation. However, IL-2 production can be observed only in the CD4+CD8- T cell subset. These results clearly contrast data obtained with purified CD4-CD8+ T cells, which are able to proliferate when the CD3-Ti complex is activated in the presence of APC. The data presented in the present study demonstrate that a simplified model for T cell activation and clonal expansion of the two major T cell subsets involve only the CD3-Ti complex and the CD2 Ag. Under conditions where the activation signals for the T cells are restricted only to the activation of CD3-Ti and CD2, the CD4+CD8- T cells respond with IL-2 production and expression of high affinity IL-2R, whereas the CD4-CD8+ T cell subset depends on exogenous IL-2 provided by the CD4+CD8- cells. These data do not, however, exclude an involvement of other cell-surface signals for regulation and control of T cell activation and T cell effector functions.     

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.     

CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.     

A novel costimulation pathway via the 4C8 antigen for the induction of CD4+ regulatory T cells

CD4(+)CD25(+) regulatory T (Treg) cells naturally occur in mice and humans, and similar Treg cells can be induced in vivo and in vitro. However, the molecular mechanisms that mediate the generation of these Treg cell populations remain unknown. We previously described anti-4C8 mAbs that inhibit the postadhesive transendothelial migration of T cells through human endothelial cell monolayers. We demonstrate in this work that Treg cells are induced by costimulation of CD4(+) T cells with anti-CD3 plus anti-4C8. The costimulation induced full activation of CD4(+) T cells with high levels of IL-2 production and cellular expansion that were comparable to those obtained on costimulation by CD28. However, upon restimulation, 4C8-costimulated cells produced high levels of IL-10 but no IL-2 or IL-4, and maintained high expression levels of CD25 and intracellular CD152, as compared to CD28-costimulated cells. The former cells showed hyporesponsiveness to anti-CD3 stimulation and suppressed the activation of bystander T cells depending on cell contact but not IL-10 or TGF-beta. The suppressor cells developed from CD4(+)CD25(-)CD45RO(+) cells. The results suggest that 4C8 costimulation induces the generation of Treg cells that share phenotypic and functional features with CD4(+)CD25(+) T cells, and that CD25(-) memory T cells may differentiate into certain Treg cell subsets in the periphery.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

IL-15 promotes survival but not effector function differentiation of CD8+ TCRalphabeta+ intestinal intraepithelial lymphocytes

CD8 single-positive cells, including CD8alphaalpha+ and CD8alphabeta+ subsets, constitute the majority of TCRalphabeta+ intestinal intraepithelial lymphocytes (alphabeta iIEL) in mice. CD8+ alphabeta iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ alphabeta iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ alphabeta iIEL subsets in vitro. In contrast to the death of approximately 60% of both CD8alphaalpha+ and CD8alphabeta+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8alphaalpha+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8alphaalpha+ alphabeta iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15's prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-gamma production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ alphabeta iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8alphaalpha+ alphabeta iIEL upon Ag stimulation.     

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.     

Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.     

Prolonged TCR/CD28 engagement drives IL-2-independent T cell clonal expansion through signaling mediated by the mammalian target of rapamycin

Proliferation of Ag-specific T cells is central to the development of protective immunity. The concomitant stimulation of the TCR and CD28 programs resting T cells to IL-2-driven clonal expansion. We report that a prolonged occupancy of the TCR and CD28 bypasses the need for autocrine IL-2 secretion and sustains IL-2-independent lymphocyte proliferation. In contrast, a short engagement of the TCR and CD28 only drives the expansion of cells capable of IL-2 production. TCR/CD28- and IL-2-driven proliferation revealed a different requirement for PI3K and for the mammalian target of rapamycin (mTOR). Thus, both PI3K and mTOR activities were needed for T cells to proliferate to TCR/CD28-initiated stimuli and for optimal cyclin E expression. In contrast, either PI3K or mTOR were sufficient for IL-2-driven cell proliferation as they independently mediated cyclin E induction. Interestingly, rapamycin delayed cell cycle entry of IL-2-sufficient T cells, but did not prevent their expansion. Together, our findings indicate that the TCR, CD28, and IL-2 independently control T cell proliferation via distinct signaling pathways involving PI3K and mTOR. These data suggest that Ag persistence and the availability of costimulatory signals and of autocrine and paracrine growth factors individually shape T lymphocyte expansion in vivo.     

IL-15 and cognate antigen successfully expand de novo-induced human antigen-specific regulatory CD4+ T cells that require antigen-specific activation for suppression

An important prerequisite in using regulatory T cells for immunotherapy is their ex vivo expansion without loss of suppressor function. Human anergic regulatory T cells are expandable by Ag-specific stimulation in the presence of IL-2. IL-15, like IL-2, is a T cell growth factor that, in contrast to IL-2, stimulates survival of T cells. In this study, we examined whether IL-15 could be exploited as a superior growth factor of human CD4(+) anergic regulatory T cells that were generated by costimulation blockade. Next, IL-15, as compared with IL-2, was investigated with respect to expansion and function of these regulatory T cells. Optimal expansion required cognate allogeneic stimulation in the presence of exogenous IL-15. IL-15 resulted in enhanced survival that was paralleled by an increased number of Bcl-2-expressing cells. Moreover, IL-15 induced a distinct type of anergy characterized by hyperreactivity to IL-15, resulting in improved expansion. This is likely attributed to increased propensity of these cells to up-regulate both alpha- and gamma-chains of the IL-2 and IL-15 receptor. Notably, IL-15-expanded regulatory CD4(+) T cells suppressed both naive and memory T cells in a superior way. Immunosuppression required alloantigen-specific stimulation and appeared gamma-irradiation resistant and independent of IL-10, TGFbeta, or CTLA-4 interactions. These regulatory T cells were stable suppressors, mediating bystander suppression upon TCR stimulation, but leaving recall responses unaffected in the absence of cognate Ag. Finally, human naturally occurring regulatory CD4(+)CD25(+) T cells appeared important in generating regulatory T cells by costimulation blockade. In conclusion, IL-15-expanded, de novo-induced human anergic regulatory CD4(+) T cells are of interest in Ag-specific immunotherapy.