Protective Mechanisms Induced by a Japanese Encephalitis Virus DNA Vaccine: Requirement for Antibody but Not CD8+ Cytotoxic T-Cell Responses

We have previously shown that a plasmid (pE) encoding the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection against a lethal viral challenge. In the present study, we used adoptive transfer experiments and gene knockout mice to demonstrate that the DNA-induced E-specific antibody alone can confer protection in the absence of cytotoxic T-lymphocyte (CTL) functions. Plasmid pE administered by either intramuscular or gene gun injection produced significant E-specific antibodies, helper T (Th)-cell proliferative responses, and CTL activities. Animals receiving suboptimal DNA vaccination produced low titers of anti-E antibodies and were only partially or not protected from viral challenge, indicating a strong correlation between anti-E antibodies and the protective capacity. This observation was confirmed by adoptive transfer experiments. Intravenous transfer of E-specific antisera but not crude or T-cell-enriched immune splenocytes to sublethally irradiated hosts conferred protection against a lethal JEV challenge. Furthermore, experiments with gene knockout mice showed that DNA vaccination did not induce anti-E titers and protective immunity in Igmu(-/-) and I-Abeta(-/-) mice, whereas in CD8alpha(-/-) mice the pE-induced antibody titers and protective rate were comparable to those produced in the wild-type mice. Taken together, these results demonstrate that the anti-E antibody is the most critical protective component in this JEV challenge model and that production of anti-E antibody by pE DNA vaccine is dependent on the presence of CD4(+) T cells but independent of CD8(+) T cells.     

Rapid and Irreversible CD4+ T-Cell Depletion Induced by the Highly Pathogenic Simian/Human Immunodeficiency Virus SHIVDH12R Is Systemic and Synchronous

Highly pathogenic simian/human immunodeficiency virus chimeric viruses are known to induce a rapid, irreversible depletion of CD4+ T lymphocytes in the peripheral blood of acutely infected macaque monkeys. To more fully assess the systemic effects of this primary virus infection, specimens were collected serially between days 3 and 21 postinfection from variety of lymphoid tissues (lymph nodes, thymus, and spleen) and gastrointestinal tract and examined by DNA and RNA PCR, in situ hybridization, and immunohistochemical assays. In addition, the lymphoid tissues were evaluated by fluorescence-activated cell sorting. Virus infection was initially detected by DNA PCR on day 3 postinfection in lymph node samples and peaked on day 10 in the T-lymphocyte-rich areas of this tissue. CD4+ T-cell levels remained stable through day 10 in several lymphoid tissue specimens examined but fell precipitously between days 10 and 21. In situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed the accumulation of apoptotic cells during the second week of infection in both lymph nodes and thymus, which colocalized, to a large extent, to sites of both virus replication and CD4+ T-lymphocyte loss.     

CD4+-T-Cell and CD20+-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques

Simian-human immunodeficiency virus 89.6PD (SHIV_89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4⁺ T cells, sustained decreases in CD20⁺ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4⁺ T cells; showed increases in circulating CD20⁺ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P ≤ 0.002), B- and T-cell losses (P ≤ 0.013), and failure to seroconvert (P ≤ 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.     

Costimulation of Naive CD8+ Lymphocytes Induces CD4 Expression and Allows Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus type 1 (HIV-1) infection requires cell surface expression of CD4. Costimulation of CD8⁺/CD4⁻ T lymphocytes by anti-CD3 and anti-CD28 antibodies or by allogeneic dendritic cells induced expression of CD4 and rendered these CD8 cells susceptible to HIV-1 infection. Naive CD45RA⁺ cells responded with greater expression of CD4 than did CD45RO⁺ cells. CD8⁺ lymphocytes derived from fetal or newborn sources exhibited a greater tendency to express CD4, consistent with their naive states. This mechanism of infection suggests HIV-induced perturbation of the CD8 arm of the immune response and could explain the generally rapid disease progression seen in HIV-infected children.     

Residual Viral Replication during Antiretroviral Therapy Boosts Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses in Subjects Treated Early after Infection

Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+ T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+ T cells in patients treated during early (n = 31) or chronic (n = 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+ T-cell response. HIV-1-specific CD8+ T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia.     

CD8+ T-Cell Epitope Mapping for Pneumonia Virus of Mice in H-2b Mice

The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8⁺ T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8⁺ T-cell epitopes restricted by H-2D^b and/or H-2K^b were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8⁺ T cells to PVM pathogenesis.     

Both Memory and CD45RA+/CD62L+ Naive CD4+ T Cells Are Infected in Human Immunodeficiency Virus Type 1-Infected Individuals

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.     

The Simian Immunodeficiency Virus Δnef Vaccine, after Application to the Tonsils of Rhesus Macaques, Replicates Primarily within CD4+ T Cells and Elicits a Local Perforin-Positive CD8+ T-Cell Response

Deletion of the nef gene from simian immunodeficiency virus (SIV) strain SIVmac239 yields a virus that undergoes attenuated growth in rhesus macaques and offers substantial protection against a subsequent challenge with some SIV wild-type viruses. We used a recently described model to identify sites in which the SIVDeltanef vaccine strain replicates and elicits immunity in vivo. A high dose of SIVDeltanef was applied to the palatine and lingual tonsils, where it replicated vigorously in this portal of entry at 7 days. Within 2 weeks, the virus had spread and was replicating actively in axillary lymph nodes, primarily in extrafollicular T-cell-rich regions but also in germinal centers. At this time, large numbers of perforin-positive cells, both CD8(+) T cells and CD3-negative presumptive natural killer cells, were found in the tonsil and axillary lymph nodes. The number of infected cells and perforin-positive cells then fell. When autopsy studies were carried out at 26 weeks, only 1 to 3 cells hybridized for viral RNA per section of lymphoid tissue. Nevertheless, infected cells were detected chronically in most lymphoid organs, where the titers of infectious virus could exceed by a log or more the titers in blood. Immunocytochemical labeling at the early active stages of infection showed that cells expressing SIVDeltanef RNA were CD4(+) T lymphocytes. A majority of infected cells were not in the active cell cycle, since 60 to 70% of the RNA-positive cells in tissue sections lacked the Ki-67 cell cycle antigen, and both Ki-67-positive and -negative cells had similar grain counts for viral RNA. Macrophages and dendritic cells, identified with a panel of monoclonal antibodies to these cells, were rarely infected. We conclude that the attenuated growth and protection observed with the SIVDeltanef vaccine strain does not require that the virus shift its characteristic site of replication, the CD4(+) T lymphocyte. In fact, this immunodeficiency virus can replicate actively in CD4(+) T cells prior to being contained by the host, at least in part by a strong killer cell response that is generated acutely in the infected lymph nodes.     

Genetic Regulation of Long-Term Nonprogression in E-55+ Murine Leukemia Virus Infection in Mice

Certain inbred mouse strains display progression to lymphoma development after infection with E-55+ murine leukemia virus (E-55+ MuLV), while others demonstrate long-term nonprogression. This difference in disease progression occurs despite the fact that E-55+ MuLV causes persistent infection in both immunocompetent BALB/c-H-2(k) (BALB.K) progressor (P) and C57BL/10-H-2(k) (B10.BR) long-term nonprogressor (LTNP) mice. In contrast to immunocompetent mice, immunosuppressed mice from both P and LTNP strains develop lymphomas about 2 months after infection, indicating that the LTNP phenotype is determined by the immune response of the infected mouse. In this study, we used bone marrow chimeras to demonstrate that the LTNP phenotype is associated with the genotype of donor bone marrow and not the recipient microenvironment. In addition, we have mapped a genetic locus that may be responsible for the LTNP trait. Microsatellite-based linkage analysis demonstrated that a non-major histocompatibility complex gene on chromosome 15 regulates long-term survival and is located in the same region as the Rfv3 gene. Rfv3 is involved in recovery from Friend virus-induced leukemia and has been demonstrated to regulate neutralizing virus antibody titers. In our studies, however, both P and LTNP strains produce similar titers of neutralizing and cytotoxic anti-E-55+ MuLV. Therefore, while it is possible that Rfv3 influences the course of E-55+ MuLV infection, it is more likely that the LTNP phenotype in E-55+ MuLV-infected mice is regulated by a different, closely linked gene.     

CD4+ T-Lymphocyte Depletion in Human Lymphoid Tissue Ex Vivo Is Not Induced by Noninfectious Human Immunodeficiency Virus Type 1 Virions

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4⁺ T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4⁺ T-cell depletion, consistent with mechanisms requiring productive infection.     

Two Overlapping Subdominant Epitopes Identified by DNA Immunization Induce Protective CD8+ T-Cell Populations with Differing Cytolytic Activities

Subdominant CD8(+) T-cell responses contribute to control of several viral infections and to vaccine-induced immunity. Here, using the lymphocytic choriomeningitis virus model, we demonstrate that subdominant epitopes can be more reliably identified by DNA immunization than by other methods, permitting the identification, in the virus nucleoprotein, of two overlapping subdominant epitopes: one presented by L(d) and the other presented by K(d). This subdominant sequence confers immunity as effective as that induced by the dominant epitope, against which >90% of the antiviral CD8(+) T cells are normally directed. We compare the kinetics of the dominant and subdominant responses after vaccination with those following subsequent viral infection. The dominant CD8(+) response expands more rapidly than the subdominant responses, but after virus infection is cleared, mice which had been immunized with the "dominant" vaccine have a pool of memory T cells focused almost entirely upon the dominant epitope. In contrast, after virus infection, mice which had been immunized with the "subdominant" vaccine retain both dominant and subdominant memory cells. During the acute phase of the immune response, the acquisition of cytokine responsiveness by subdominant CD8(+) T cells precedes their development of lytic activity. Furthermore, in both dominant and subdominant populations, lytic activity declines more rapidly than cytokine responsiveness. Thus, the lysis(low)-cytokine(competent) phenotype associated with most memory CD8(+) T cells appears to develop soon after antigen clearance. Finally, lytic activity differs among CD8(+) T-cell populations with different epitope specificities, suggesting that vaccines can be designed to selectively induce CD8(+) T cells with distinct functional attributes.     

How Does Cross-Reactive Stimulation Affect the Longevity of CD8+ T Cell Memory?

Immunological memory—the ability to “remember” previously encountered pathogens and respond faster upon re-exposure is a central feature of the immune response in vertebrates. The cross-reactive stimulation hypothesis for the maintenance of memory proposes that memory cells specific for a given pathogen are maintained by cross-reactive stimulation following infections with other (unrelated) pathogens. We use mathematical models to examine the cross-reactive stimulation hypothesis. We find that: (i) the direct boosting of cross-reactive lineages only provides a very small increase in the average longevity of immunological memory; (ii) the expansion of cross-reactive lineages can indirectly increase the longevity of memory by reducing the magnitude of expansion of new naive lineages which occupy space in the memory compartment and are responsible for the decline in memory; (iii) cross-reactive stimulation results in variation in the rates of decline of different lineages of memory cells and enrichment of memory cell population for cells that are cross-reactive for the pathogens to which the individual has been exposed.     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

CD40 Ligand-Mediated Interactions Are Involved in the Generation of Memory CD8+ Cytotoxic T Lymphocytes (CTL) but Are Not Required for the Maintenance of CTL Memory following Virus Infection

CD8⁺ cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8⁺ T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8⁺ CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8⁺ CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8⁺ CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8⁺ T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8⁺ cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4⁺ T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8⁺ memory CTLp, the probable nature of which is discussed.     

Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+ Cytotoxic T-Cell Epitopes

Compelling evidence now suggests that alphabeta CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.     

Expression of the Mucosal Homing Receptor α4β7 Correlates with the Ability of CD8+ Memory T Cells To Clear Rotavirus Infection

The integrin α₄β₇ plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with α₄β₇ expression. α₄β₇^hi memory phenotype (CD44^hi), α₄β₇⁻ memory phenotype, and presumptively naive (CD44^lo) CD8⁺ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. α₄β₇^hi memory phenotype CD8⁺ cells were highly efficient at clearing rotavirus infection, α₄β₇⁻ memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44^lo cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor α₄β₇.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.