TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guérin infection via augmentation of NK and T cytotoxic 1 responses.

To investigate the immunomodulating effects of IL-15 in vivo on mycobacterial infection, we used IL-15-transgenic (Tg) mice, which were recently constructed with cDNA-encoding secretable isoform of IL-15 precursor protein under the control of a MHC class I promoter. The IL-15-Tg mice exhibited resistance against infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), as assessed by bacteria growth. IFN-gamma level in serum was significantly higher in IL-15-Tg mice than in non-Tg mice after BCG infection. NK cells were remarkably increased, and Ag-specific T cytotoxic 1 response mediated by CD8+ T cells producing IFN-gamma was significantly augmented in the IL-15-Tg mice following BCG infection. Neutralization of endogenous IFN-gamma by in vivo administration of anti-IFN-gamma mAb deteriorated the clearance of the bacteria. Depletion of of NK cells or CD8+ T cells by in vivo administration of anti-asialo-GM(1) Ab or anti-CD8 mAb hampered the exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced protection against BCG infection via augmentation of NK and T cytotoxic 1 responses.     

Infiltration of a mesothelioma by IFN-gamma-producing cells and tumor rejection after depletion of regulatory T cells

Depletion of CD4+CD25+Foxp3+ regulatory T cells (CD25+ T(reg)) with an anti-CD25 Ab results in immune-mediated rejection of tolerogenic solid tumors. In this study, we have examined the immune response to a mesothelioma tumor in mice after depletion of CD25+ cells to elucidate the cellular mechanisms of CD25+ T(reg), a subject over which there is currently much conjecture. Tumor rejection was found to be primarily due to the action of CD8+ T cells, although CD4+ cells appeared to play some role. Depletion of CD25+ cells resulted in an accumulation in tumor tissue of CD4+ and CD8+ T cells and NK cells that were producing the potent antitumor cytokine IFN-gamma. Invasion of tumors by CD8+ T cells was partially dependent on the presence of CD4+ T cells. Although a significant increase in the proliferation and number of tumor-specific CD8+ T cells was observed in lymph nodes draining the tumor of anti-CD25-treated mice, this effect was relatively modest compared with the large increase in IFN-gamma-producing T cells found in tumor tissue, which suggests that the migration of T cells into tumor tissue may also have been altered. Depletion of CD25+ cells did not appear to modulate antitumor CTL activity on a per cell basis. Our data suggests that CD25+ T(reg) limit the accumulation of activated T cells producing IFN-gamma in the tumor tissue and, to a lesser extent, activation and/or rate of mitosis of tumor-specific T cells in lymph nodes.     

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.     

NK cell-like behavior of Valpha14i NK T cells during MCMV infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Valpha14 invariant natural killer T cell response to MCMV has not been examined. We found that Valpha14i NK T cells become activated and produce significant levels of IFN-gamma, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Valpha14i NK T cells into MCMV-infected CD1d(-/-) mice demonstrate that CD1d is dispensable for Valpha14i NK T cell activation. In contrast, both IFN-alpha/beta and IL-12 are required for optimal activation. Valpha14i NK T cell-derived IFN-gamma is partially dependent on IFN-alpha/beta but highly dependent on IL-12. Valpha14i NK T cells contribute to the immune response to MCMV and amplify NK cell-derived IFN-gamma. Importantly, mortality is increased in CD1d(-/-) mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Valpha14i NK T cells that act as effector T cells during bacterial infection, but have NK cell-like behavior during the innate immune response to MCMV infection.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.     

Mononuclear phagocyte-derived IL-10 suppresses the innate IL-12/IFN-gamma axis in lung-challenged aged mice

Previously, we reported that IL-10-producing mononuclear phagocytes increase in lungs of aged mice, causing impaired innate cytokine expression. Since dendritic cells (DCs) contribute to innate NK cell and adaptive T cell immunity, we tested the hypothesis that age-related IL-10 might influence DC function with effects on NK and T cell activation. The results showed that DC recruitment to sites of lung inflammation was normal in aged mice (>20 mo). However, IFN-gamma-producing NK cells in LPS-challenged lungs were decreased in aged as compared with young mice, which was associated with increased IL-10(+)CD11b(+)Gr-1(low)CD11c(-) cells consistent with mononuclear phagocytes. In vivo or in vitro blockade of IL-10 signaling restored IFN-gamma-producing NK cells. This restoration was reversed by IL-12 neutralization, indicating that IL-10 suppressed sources of IL-12 in aged mice. To probe DC function in adaptive immunity, we transferred young naive OVA-specific TCR transgenic T cells to old mice. Following challenge with OVA plus LPS, Ag presentation in the context of MHC-I and MHC-II occurred with similar kinetics and intensity in draining lymph nodes of young and old recipients as measured by proliferation. Despite this, aged hosts displayed impaired induction of IFN-gamma(+)CD4(+), but not IFN-gamma(+)CD8(+), effector T cells. Blockade of IL-10 signaling reversed age-associated defects. These studies indicate that the innate IL-12/IFN-gamma axis is not intrinsically defective in lungs of aged mice, but is rather suppressed by enhanced production of mononuclear phagocyte-derived IL-10. Our data identify a novel mechanism of age-associated immune deficiency.     

Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice

In this study we evaluated the ability of activated intrahepatic APCs to inhibit hepatitis B virus (HBV) replication in transgenic mice. Intrahepatic APCs were activated by administration of an anti-CD40 agonistic mAb (alphaCD40). We showed that a single i.v. injection of alphaCD40 was sufficient to inhibit HBV replication noncytopathically by a process associated with the recruitment of dendritic cells, macrophages, T cells, and NK cells into the liver and the induction of inflammatory cytokines. The antiviral effect depended on the production of IL-12 and TNF-alpha by activated APCs; however, it was mediated primarily by IFN-gamma produced by NK cells, and possibly T cells, that were activated by IL-12. Collectively, these results suggest that activated APCs can directly produce antiviral cytokines (IL-12, TNF-alpha) and trigger the production of other cytokines (i.e., IFN-gamma) by other cells (e.g., NK cells and T cells) that do not express CD40. These results provide insight into a hitherto unsuspected antiviral function of intrahepatic APCs, and they suggest that therapeutic activation of APCs may represent a new strategy for the treatment of chronic HBV infection.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

Murine plasmacytoid dendritic cells produce IFN-gamma upon IL-4 stimulation

IL-4 plays a key role in inducing IL-4 production in CD4+ T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN-gamma production in B220+ plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN-gamma upon IL-4 stimulation, we found that PDCs were a major IFN-gamma-producing cell upon IL-4 stimulation in wild-type and Rag-2-/- splenocytes. Isolated PDCs, but not CD11b+ DCs or CD8+ DCs, produced IFN-gamma upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN-gamma production induced by IL-4 administration. We also found that IL-4 induced IFN-gamma production, but not IL-12 or IFN-alpha production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN-gamma production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN-alpha production. However, IL-4 did not induce IFN-gamma production in Stat6-/- PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN-gamma. Furthermore, IL-4 did not induce IFN-gamma production in Stat4-/- PDCs. These results indicate that PDCs preferentially produce IFN-gamma upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.     

Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.