Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.     

Dynamic regulation of IFN-gamma signaling in antigen-specific CD8+ T cells responding to infection

IFN-gamma plays a critical role in the CD8(+) T cell response to infection, but when and if this cytokine directly signals CD8(+) T cells during an immune response is unknown. We show that naive Ag-specific CD8(+) T cells receive IFN-gamma signals within 12 h after in vivo infection with Listeria monocytogenes and then become unresponsive to IFN-gamma throughout the ensuing Ag-driven expansion phase. Ag-specific CD8(+) T cells regain partial IFN-gamma responsiveness throughout the contraction phase, whereas the memory pool exhibits uniform, but reduced, responsiveness that is also modulated during the secondary response. The responsiveness of Ag-specific CD8(+) T cells to IFN-gamma correlated with modulation in the expression of IFN-gammaR2, but not with IFN-gammaR1 or suppressor of cytokine signaling-1. This dynamic regulation suggests that early IFN-gamma signals participate in regulation of the primary CD8(+) T cell response program, but that evading or minimizing IFN-gamma signals during expansion and the memory phase may contribute to appropriate regulation of the CD8(+) T cell response.     

Direct interferon-gamma signaling dramatically enhances CD4+ and CD8+ T cell memory

Studies in IFN-gamma-deficient mice suggest that the delivery of IFN-gamma to CD8(+) T cells early in virus infection programs their eventual contraction, thereby reducing the abundance of CD8(+) memory T cells. In this study, we show that such mice fail to completely eliminate virus infection and that, when evaluated without the confounding factor of persisting Ag, both CD4(+) and CD8(+) T cells undergo profound contraction when they are unable to receive IFN-gamma signals. Furthermore, the abundance of CD4(+) and CD8(+) memory cells that express the IFN-gamma receptor is approximately 100-fold higher than cells lacking this molecule. Thus, direct IFN-gamma signaling is not required for T cell contraction during virus infection, and it enhances, rather than suppresses, the development of virus-specific CD4(+) and CD8(+) T cell memory.     

The induction of HIV Gag-specific CD8+ T cells in the spleen and gut-associated lymphoid tissue by parenteral or mucosal immunization with recombinant Listeria monocytogenes HIV Gag

The induction of mucosal immunity is crucial in controlling viral replication during HIV infection. In this study we compare the ability of a recombinant Listeria monocytogenes that expresses and secretes the HIV Ag Gag to induce CD8(+) T cells against this Ag in the spleen, mesenteric lymph nodes, and Peyer's patches and the ability to provide effector Gag-specific CD8(+) T cells to the lamina propria after i.v., oral, or rectal administration of the vaccine. The levels of Ag-specific CD8(+)-activated T cells were measured ex vivo using intracellular cytokine staining for IFN-gamma and H-2K(d) Gag peptide tetramer staining. We found that all routes of immunization induced Gag-specific CD8(+) T cells in the spleen. After secondary infection, we observed substantial increases in splenic levels of CD8(+) T cells, and levels of Gag-specific cells were similar to those against listeriolysin O, the immunodominant Ag of L. monocytogenes. Both primary and secondary oral immunization resulted in abundant Gag-specific CD8(+)-activated T cells in the lamina propria that constituted approximately 35% of the CD8 compartment. However, significant levels of Gag and listeriolysin O-specific CD8(+) T cells were observed in mucosal lymphoid tissue only after two immunizations, perhaps because they had already entered the lamina propria compartment after a single immunization. In the context of HIV, a mucosally administered vaccine seems best calculated to prompt an immune response that is capable of preventing infection. The data presented in this report demonstrate that mucosally administered Listeria can prompt such a response and that booster doses can maintain this response.     

Stimulation by soluble CD70 promotes strong primary and secondary CD8+ cytotoxic T cell responses in vivo

Identification of the signals required for optimal differentiation of naive CD8(+) T cells into effector and memory cells is critical for the design of effective vaccines. In this study we demonstrate that CD27 stimulation by soluble CD70 considerably enhances the magnitude and quality of the CD8(+) T cell response. Stimulation with soluble CD70 in the presence of Ag significantly enhanced the proliferation of CD8(+) T cells and their ability to produce IL-2 and IFN-gamma in vitro. Administration of Ag and soluble CD70 resulted in a massive (>300-fold) expansion of Ag-specific CD8(+) T cells in vivo, which was due to the enhanced proliferation and survival of activated T cells. In mice that received Ag and soluble CD70, CD8(+) T cells developed into effectors with direct ex vivo cytotoxicity. Furthermore, unlike peptide immunization, which resulted in a diminished response after rechallenge, CD27 stimulation during the primary challenge evoked a strong secondary response upon rechallenge with the antigenic peptide. Thus, in addition to increasing the frequency of primed Ag-specific T cells, CD27 signaling during the primary response instills a program of differentiation that allows CD8(+) T cells to overcome a state of unresponsiveness. Taken together these results demonstrate that soluble CD70 has potent in vivo adjuvant effects for CD8(+) T cell responses.     

The central memory CD4+ T cell population generated during Leishmania major infection requires IL-12 to produce IFN-gamma

Central memory CD4(+) T cells provide a pool of lymph node-homing, Ag-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of central memory CD4(+) T cells in Leishmania major-infected mice that were capable of mediating immunity to a secondary infection. In this study, we show that the Leishmania-specific central memory CD4(+) T cells require IL-12 to produce IFN-gamma, demonstrating that this population needs additional signals to develop into Th1 cells. In contrast, effector cells isolated from immune mice produced IFN-gamma in vitro or in vivo in the absence of IL-12. In addition, we found that when central memory CD4(+) T cells were adoptively transferred into IL-12-deficient hosts, many of the cells became IL-4 producers. These studies indicate that the central memory CD4(+) T cell population generated during L. major infection is capable of developing into either Th1 or Th2 effectors. Thus, continued IL-12 production may be required to ensure the development of Th1 cells from this central memory T cell pool, a finding that has direct relevance to the design of vaccines dependent upon central memory CD4(+) T cells.     

Functional heterogeneity of cytokines and cytolytic effector molecules in human CD8+ T lymphocytes.

CD8(+) T cells use a number of effector mechanisms to protect the host against infection. We have studied human CD8(+) T cells specific for CMV pp65(495-503) epitope, or for staphylococcal enterotoxin B, for the expression patterns of five cytokines and cytolytic effector molecules before and after antigenic stimulation. Ex vivo, the cytolytic molecule granzyme B was detected in a majority of circulating CMV-specific CD8(+) T cells, whereas perforin was rarely expressed. Both were highly expressed after Ag-specific activation accompanied by CD45RO up-regulation. TNF-alpha, IFN gamma, and IL-2 were sequentially acquired on recognition of Ag, but surprisingly, only around half of the CMV-specific CD8(+) T cells responded to antigenic stimuli with production of any cytokine measured. A dominant population coexpressed TNF-alpha and IFN-gamma, and cells expressing TNF-alpha only, IFN-gamma only, or all three cytokines together also occurred at lower but clearly detectable frequencies. Interestingly, perforin expression and production of IFN-gamma and TNF-alpha in CD8(+) T cells responding to staphylococcal enterotoxin B appeared to be largely segregated, and no IL-2 was detected in perforin-positive cells. Together, these data indicate that human CD8(+) T cells can be functionally segregated in vivo and have implications for the understanding of human CD8(+) T cell differentiation and specialization and regulation of effector mechanisms.     

T cells undergo rapid ON/OFF but not ON/OFF/ON cycling of cytokine production in response to antigen

Inflammatory cytokines such as IFN-gamma and TNF produced by Ag-stimulated CD4(+) and CD8(+) T cells are important in defense against microbial infection. However, production of these cytokines must be tightly regulated to prevent immunopathology. Previous studies, conducted with BALB/c mice, have suggested that 1) CD8(+) T cells maintain IFN-gamma production but transiently produce TNF in the continued presence of Ag and 2) lymphocytic choriomeningitis virus-specific and in vitro-propagated effector CD8(+) T cells could rapidly cycle IFN-gamma production ON/OFF/ON in response to Ag exposure, removal, and re-exposure. In contrast with CD8(+) T cells, our results show that Listeria monocytogenes-specific CD4(+) T cells from C57BL/6 mice rapidly initiate (ON cycling) and maintain production of both IFN-gamma and TNF in the continued presence of Ag. Upon Ag removal, production of both cytokines rapidly ceases (OFF cycling). However, if the initial stimulation was maximal, Ag-specific CD4(+) T cells were unable to reinitiate cytokine production after a second Ag exposure. Furthermore, L. monocytogenes-specific CD8(+) T cells in the same mice and lymphocytic choriomeningitis virus-specific CD8(+) T cells in BALB/c mice also underwent ON/OFF cycling, but if the initial Ag stimulus was maximal, they could not produce IFN-gamma after Ag re-exposure. As the initial Ag dose was reduced, the number of cells producing cytokine in response to the second Ag exposure exhibited a corresponding increase. However, T cells that were marked for IFN-gamma secretion during the first stimulation did not contribute cytokine production during the second stimulation. Thus, T cells are not able to undergo rapid ON/OFF/ON cytokine cycling in vitro in response to Ag.     

Role of CD28 for the generation and expansion of antigen-specific CD8(+) T lymphocytes during infection with Listeria monocytogenes.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity. Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice. In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions. Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis. However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.     

Induction and visualization of mucosal memory CD8 T cells following systemic virus infection.

Whether CD8 T cell memory exists outside secondary lymphoid organs is unclear. Using an adoptive transfer system that enables tracking of OVA-specific CD8 T cells, we explored the antigenic requirements for inducing CD8 T cell memory and identified intestinal mucosa memory cells. Although systemic immunization with soluble OVA induced clonal expansion, memory CD8 cells were not produced. In contrast, infection with virus-encoding OVA induced memory CD8 cells in the periphery and the lamina propria and intraepithelial compartments of the intestinal mucosa. Mucosal memory cells expressed a distinct array of adhesion molecules as compared with secondary lymphoid memory cells, suggesting that there may be separate mucosal and systemic memory pools. Mucosal CD8 memory cells rapidly produced IFN-gamma after Ag stimulation. Reactivation of memory cells by Ag feeding resulted in increased cell size and up-regulation of CD28 and CD11c. CD8 mucosal memory cells exhibited ex vivo lytic activity that was up-regulated dramatically following Ag reencounter in vivo. Interestingly, reactivation of memory cells did not require CD28-mediated costimulation. The ability of the intestinal mucosa to maintain CD8 memory cells provides a potential mechanism for effective mucosal vaccination.     

Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo

Recent studies have shown that CD4(+) T cell help is required for the generation of memory CD8(+) T cells that can proliferate and differentiate into effector cells on Ag restimulation. The importance of help for primary CD8(+) T cell responses remains controversial. It has been suggested that help is not required for the initial proliferation and differentiation of CD8(+) T cells in vivo and that classical models of helper-dependent responses describe impaired secondary responses to Ag in vitro. We have measured primary CD8(+) T cell responses to peptide-pulsed dendritic cells in mice by cytokine ELISPOT and tetramer staining. No responses were detected in the absence of help, either when normal dendritic cells were injected into MHC II-deficient mice or when MHC II-deficient dendritic cells were injected into normal mice. Thus, the primary in vivo CD8(+) T cell response depends absolutely on help from CD4(+) T cells in our experimental system.     

Cutting edge: OFF cycling of TNF production by antigen-specific CD8+ T cells is antigen independent

Although they are known for their capacity to kill infected cells, Ag-specific CD8(+) T cells elaborate other effector mechanisms, including TNF and IFN-gamma, that contribute to defense against infection. Ag-specific CD8(+) T cells rapidly turn ON and turn OFF IFN-gamma production in direct response to Ag contact, presumably to minimize the potential immunopathology that could result from inappropriate secretion of this inflammatory mediator. In this study, we show, using in vitro propagated and directly ex vivo-analyzed Ag-specific CD8(+) T cells, that in contrast to Ag-dependent ON/OFF cycling of IFN-gamma production, the cessation of TNF production by the same IFN-gamma producing cells is rapid and Ag independent.     

Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens

Substantial CD8(+) T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP(118-126)) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8(+) T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8(+) T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thus, neutrophils not only provide rapid innate defense against infection, but also contribute to shaping the specificity and breadth of the CD8(+) T cell response. In addition, cross-presentation of bacterial Ags from neutrophils may explain how CD8(+) T cell responses are generated against Ags from extracellular bacterial pathogens.     

Differential regulation of primary and secondary CD8+ T cells in the central nervous system

T cell accumulation and effector function following CNS infection is limited by a paucity of Ag presentation and inhibitory factors characteristic of the CNS environment. Differential susceptibilities of primary and recall CD8+ T cell responses to the inhibitory CNS environment were monitored in naive and CD8+ T cell-immune mice challenged with a neurotropic coronavirus. Accelerated virus clearance and limited spread in immunized mice was associated with a rapid and increased CNS influx of virus-specific secondary CD8+ T cells. CNS-derived secondary CD8+ T cells exhibited increased cytolytic activity and IFN-gamma expression per cell compared with primary CD8+ T cells. However, both Ag-specific primary and secondary CD8+ T cells demonstrated similar contraction rates. Thus, CNS persistence of increased numbers of secondary CD8+ T cells reflected differences in the initial pool size during peak inflammation rather than enhanced survival. Unlike primary CD8+ T cells, persisting secondary CD8+ T cells retained ex vivo cytolytic activity and expressed high levels of IFN-gamma following Ag stimulation. However, both primary and secondary CD8+ T cells exhibited reduced capacity to produce TNF-alpha, differentiating them from effector memory T cells. Activation of primary and secondary CD8+ T cells in the same host using adoptive transfers confirmed similar survival, but enhanced and prolonged effector function of secondary CD8+ T cells in the CNS. These data suggest that an instructional program intrinsic to T cell differentiation, rather than Ag load or factors in the inflamed CNS, prominently regulate CD8+ T cell function.     

Partially TAP-independent protection against Listeria monocytogenes by H2-M3-restricted CD8+ T cells

Effective protection against Listeria monocytogenes requires Ag-specific CD8(+) T cells. A substantial proportion of CD8(+) T cells activated during L. monocytogenes infection of C57BL/6 mice are restricted by the MHC class Ib molecule H2-M3. In this study, an H2-M3-restricted CD8(+) T cell clone specific for a known H2-M3 epitope (fMIGWII) was generated from L. monocytogenes-infected mice. The clone was cytotoxic, produced IFN-gamma, and could mediate strong protection against L. monocytogenes when transferred to infected mice. Macrophages pulsed with heat-killed LISTERIAE: presented Ag to the clone in a TAP-independent manner. Both TAP-independent and -dependent processing occurred in vivo, as TAP-deficient mice infected with L. monocytogenes were partially protected by adoptive transfer of the clone. This is the first example of CD8(+) T cell-mediated, TAP-independent protection against a pathogen in vivo, confirming the importance of alternative MHC class I processing pathways in the antibacterial immunity.     

Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice

Classical CD4(+) and CD8(+) T cells recognize Ag presented by MHC class II (MHCII) and MHC class I (MHCI), respectively. However, our results show that CD4(-/-) mice mount a strong, readily detectable CD8(+) T cell response to MHCII-restricted epitopes after a primary bacterial or viral infection. These MHCII-restricted CD8(+)CD4(-) T cells are more similar to classical CD8(+) T cells than to CD4(+) T cells in their expression of effector functions during a primary infection, yet they also differ from MHCI-restricted CD8(+) T cells by their inability to produce high levels of the cytolytic molecule granzyme B. After resolution of a primary infection, epitope-specific MHCII-restricted T cells in CD4(-/-) mice persist for a long period of time as memory T cells. Surprisingly, upon reinfection the secondary MHCII-restricted response in CD4(-/-) mice consists mainly of CD8(-)CD4(-) T cells. In contrast to CD8(+) T cells, MHCII-restricted CD8(-)CD4(-) T cells are capable of producing IL-2 in addition to IFN-gamma and thus appear to have attributes characteristic of CD4(+) T cells rather than CD8(+) T cells. Therefore, MHCII-restricted T cells in CD4(-/-) mice do not share all phenotypic and functional characteristics with MHCI-restricted CD8(+) T cells or with MHCII-restricted CD4(+) T cells, but, rather, adopt attributes from each of these subsets. These results have implications for understanding thymic T cell selection and for elucidating the mechanisms regulating the peripheral immune response and memory differentiation.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

Cutting edge: CD8+ effector T cells reject tumors by direct antigen recognition but indirect action on host cells

CD8(+) effector T cells recognize malignant cells by monitoring their surface for the presence of tumor-derived peptides bound to MHC class I molecules. In addition, tumor-derived Ags can be cross-presented to CD8(+) effector T cells by APCs. IFN-gamma production by CD8(+) T cells is often critical for tumor rejection. However, it remained unclear whether 1) CD8(+) T cells secrete IFN-gamma in response to Ag recognition on tumor cells or APCs and 2) whether IFN-gamma mediates its antitumor effect by acting on host or tumor cells. We show in this study that CD8(+) effector T cells can reject tumors in bone marrow-chimeric mice incapable of cross-presenting Ag by bone marrow-derived APCs and that tumor rejection required host cells to express IFN-gammaR. Together, CD8(+) effector T cells recognize Ag directly on tumor cells, and this recognition is sufficient to reject tumors by IFN-gamma acting on host cells.     

Cognate memory CD4+ T cells generated with dendritic cell priming influence the expansion, trafficking, and differentiation of secondary CD8+ T cells and enhance tumor control

CD4(+) T cells are known to provide support for the activation and expansion of primary CD8(+) T cells, their subsequent differentiation, and ultimately their survival as memory cells. However, the importance of cognate memory CD4(+) T cells in the expansion of memory CD8(+) T cells after re-exposure to Ag has been not been examined in detail. Using bone marrow-derived dendritic cells pulsed with cognate or noncognate MHC class I- and class II-restricted peptides, we examined whether the presence of memory CD4(+) T cells with the same Ag specificity as memory CD8(+) T cells influenced the quantity and quality of the secondary CD8(+) T cell response. After recombinant vaccinia virus-mediated challenge, we demonstrate that, although cognate memory CD4(+) T cells are not required for activation of secondary CD8(+) T cells, their presence enhances the expansion of cognate memory CD8(+) T cells. Cognate CD4(+) T cell help results in an approximate 2-fold increase in the frequency of secondary CD8(+) T cells in secondary lymphoid tissues, and can be accounted for by enhanced proliferation in the secondary CD8(+) T cell population. In addition, cognate memory CD4(+) T cells further selectively enhance secondary CD8(+) T cell infiltration of tumor-associated peripheral tissue, and this is accompanied by increased differentiation into effector phenotype within the secondary CD8(+) T cell population. The consequence of these improvements to the magnitude and phenotype of the secondary CD8(+) T cell response is substantial increase in control of tumor outgrowth.     

Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens

The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival. To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma. IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria. IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype. The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens.