Increased loss of CCR5+ CD45RA- CD4+ T cells in CD8+ lymphocyte-depleted Simian immunodeficiency virus-infected rhesus monkeys

Previously we have shown that CD8(+) T cells are critical for containment of simian immunodeficiency virus (SIV) viremia and that rapid and profound depletion of CD4(+) T cells occurs in the intestinal tract of acutely infected macaques. To determine the impact of SIV-specific CD8(+) T-cell responses on the magnitude of the CD4(+) T-cell depletion, we investigated the effect of CD8(+) lymphocyte depletion during primary SIV infection on CD4(+) T-cell subsets and function in peripheral blood, lymph nodes, and intestinal tissues. In peripheral blood, CD8(+) lymphocyte-depletion changed the dynamics of CD4(+) T-cell loss, resulting in a more pronounced loss 2 weeks after infection, followed by a temporal rebound approximately 2 months after infection, when absolute numbers of CD4(+) T cells were restored to baseline levels. These CD4(+) T cells showed a markedly skewed phenotype, however, as there were decreased levels of memory cells in CD8(+) lymphocyte-depleted macaques compared to controls. In intestinal tissues and lymph nodes, we observed a significantly higher loss of CCR5(+) CD45RA(-) CD4(+) T cells in CD8(+) lymphocyte-depleted macaques than in controls, suggesting that these SIV-targeted CD4(+) T cells were eliminated more efficiently in CD8(+) lymphocyte-depleted animals. Also, CD8(+) lymphocyte depletion significantly affected the ability to generate SIV Gag-specific CD4(+) T-cell responses and neutralizing antibodies. These results reemphasize that SIV-specific CD8(+) T-cell responses are absolutely critical to initiate at least partial control of SIV infection.     

Gene expression in antigen-specific CD8+ T cells during viral infection

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.     

IL-15-independent proliferative renewal of memory CD8+ T cells in latent gammaherpesvirus infection

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8(+) T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8(+) T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8(+) T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8(+) T cells. Even at 140 days postinfection, the proportion of CD8(+) T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8(+) T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15(-/-) mice had a significantly higher turnover rate within the virus-specific memory CD8(+) T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15(-/-) mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8(+) T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8(+) T cells engaged in active immune surveillance of latent viral infections vs memory CD8(+) T cells found after acute viral infections.     

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.     

T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Na?ve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from na?ve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.     

Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers

Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.     

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.     

Differential regulation of primary and memory CD8 T cell immune responses by diacylglycerol kinases

The manipulation of signals downstream of the TCR can have profound consequences for T cell development, function, and homeostasis. Diacylglycerol (DAG) produced after TCR stimulation functions as a secondary messenger and mediates the signaling to Ras-MEK-Erk and NF-κB pathways in T cells. DAG kinases (DGKs) convert DAG into phosphatidic acid, resulting in termination of DAG signaling. In this study, we demonstrate that DAG metabolism by DGKs can serve a crucial function in viral clearance upon lymphocytic choriomeningitis virus infection. Ag-specific CD8(+) T cells from DGKα(-/-) and DGKζ(-/-) mice show enhanced expansion and increased cytokine production after lymphocytic choriomeningitis virus infection, yet DGK-deficient memory CD8(+) T cells exhibit impaired expansion after rechallenge. Thus, DGK activity plays opposing roles in the expansion of CD8(+) T cells during the primary and memory phases of the immune response, whereas consistently inhibiting antiviral cytokine production.     

Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo

CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.     

Role of Bim in regulating CD8+ T-cell responses during chronic viral infection

Apoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8(+) T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8(+) T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8(+) T cells by major histocompatibility complex class I tetramer staining revealed that CD8(+) D(b)NP396-404(+) T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8(+) D(b)GP33-41(+) and CD8(+) D(b)GP276-286(+) T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8(+) D(b)NP396-404(+) T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4(+) T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8(+) T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.     

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.     

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.     

Mitochondrial potential and reactive oxygen intermediates in antigen-specific CD8+ T cells during viral infection

Following many viral infections, there are large expansions of Ag-specific CD8(+) T cells. After viral clearance, mechanisms exist to ensure that the vast majority of effector cells undergo apoptosis. In studies of thymocyte apoptosis, loss of mitochondrial potential (deltapsi(m)) and excess production of reactive oxygen intermediates have been implicated as key events in cellular apoptosis. The purpose of the experiments presented in this work was to determine these parameters in Ag-specific CD8(+) T cells during a physiological response such as viral infection. Using lymphocytic choriomeningitis virus infection of mice, we found that Ag-specific CD8(+) effector T cells that had undergone recent TCR stimulation had an increased deltapsi(m). These cells also had increased levels of superoxide. As these cells progressed through the contraction of the immune response, their potential decreased, but superoxide levels remained similar to naive cells. One of the consequences of reduced mitochondrial potential is membrane permeability and subsequent caspase activation. We examined both the enzymatic activity and levels of cleaved caspase 3, an effector caspase, and could only detect increased levels in Ag-specific CD8(+) T cells on day 5 postinfection, a time point in which virus was still present. This contrasts with Ag-specific effector cells examined during the contraction phase that had no detectable caspase activity directly ex vivo. These data suggest that the apoptotic program begins earlier than previously expected on day 5, during the expansion phase.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy

The cytotoxic T-cell response in chronic hepatitis B virus (HBV) infection has been described as weak and mono- or oligospecific in comparison to the more robust virus-specific T-cell response present in resolved infection. However, chronic hepatitis B is a heterogeneous disease with markedly variable levels of virus replication and liver disease activity. Here we analyzed (both directly ex vivo and after in vitro stimulation) the HBV-specific CD8 T-cell responses against structural and nonstructural HBV proteins longitudinally in patients with different patterns of chronic infections. We found that the profiles of virus-specific CD8(+)-T-cell responses during chronic infections are highly heterogeneous and influenced more by the level of HBV replication than by the activity of liver disease. An HBV DNA load of <10(7) copies/ml appears to be the threshold below which circulating multispecific HBV-specific CD8(+) T cells are consistently detected. Furthermore, CD8(+) T cells with different specificities are differentially regulated during chronic infections. HBV core-specific CD8(+) T cells are associated with viral control, while CD8(+) T cells specific for envelope and polymerase epitopes can occasionally be found in the setting of high levels (>10(7) copies) of HBV replication. These findings have implications for the design of immunotherapy for chronic HBV infections.     

Foxp3+ regulatory T cells impede the priming of protective CD8+ T cells

T cell activation is controlled by incompletely defined opposing stimulation and suppression signals that together sustain the balance between optimal host defense against infection and peripheral tolerance. In this article, we explore the impacts of Foxp3(+) regulatory T cell (Treg) suppression in priming Ag-specific T cell activation under conditions of noninfection and infection. We find the transient ablation of Foxp3(+) Tregs unleashes the robust expansion and activation of peptide-stimulated CD8(+) T cells that provide protection against Listeria monocytogenes infection in an Ag-specific fashion. By contrast, Treg ablation had nonsignificant impacts on the CD8(+) T cell response primed by infection with recombinant L. monocytogenes. Similarly, nonrecombinant L. monocytogenes administered with peptide stimulated the expansion and activation of CD8(+) T cells that paralleled the response primed by Treg ablation. Interestingly, these adjuvant properties of L. monocytogenes did not require CD8(+) T cell stimulation by IL-12 produced in response to infection, but instead were associated with sharp reductions in Foxp3(+) Treg suppressive potency. Therefore, Foxp3(+) Tregs impose critical barriers that, when overcome naturally during infection or artificially with ablation, allow the priming of protective Ag-specific CD8(+) T cells.     

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.