Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8(+) T-cell memory

The recall of CD8(+) T-cell memory established by infecting H-2(b) mice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8(+) effector and memory T cells specific for the shared, immunodominant D(b)NP(366) epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8(+) memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2-->H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8(+) D(b)NP(366)(+) T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8(+) D(b)NP(366)(+) set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8(+) T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8(+) memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.     

The dynamics of the cellular immune response to HIV infection: implications for vaccination

Recent advances in measuring T-cell responses to viruses have led to new insights into how these T cells respond. In the acute infection there are massive CD8+ T-cell responses to both Epstein-Barr virus (EBV) and to human immunodeficiency virus (HIV). Many of these T cells are effector cells and only a minority appear to be capable of maintaining immunological memory. In persistent virus infections, high levels of antigen-specific effector cells persist. If virus does not persist, the effectors fade in number but memory is maintained and is primed to react rapidly to a new challenge. A vaccine that stimulates only T-cell responses may protect when these memory cells respond rapidly enough to generate high numbers of effectors before the infecting virus becomes established.     

Loss of naïve cells accompanies memory CD4+ T-cell depletion during long-term progression to AIDS in Simian immunodeficiency virus-infected macaques

Human immunodeficiency virus and simian immunodeficiency virus (SIV) induce a slow progressive disease, characterized by the massive loss of memory CD4+ T cells during the acute infection followed by a recovery phase in which virus replication is partially controlled. However, because the initial injury is so severe and virus production persists, the immune system eventually collapses and a symptomatic fatal disease invariably occurs. We have assessed CD4+ T-cell dynamics and disease progression in 12 SIV-infected rhesus monkeys for nearly 2 years. Three macaques exhibiting a rapid progressor phenotype experienced rapid and irreversible loss of memory, but not na?ve, CD4+ T lymphocytes from peripheral blood and secondary lymphoid tissues and died within the first 6 months of virus inoculation. In contrast, SIV-infected conventional progressor animals sustained marked but incomplete depletions of memory CD4+ T cells and continuous activation/proliferation of this T-lymphocyte subset. This was associated with a profound loss of na?ve CD4+ T cells from peripheral blood and secondary lymphoid tissues, which declined at rates that correlated with disease progression. These data suggest that the persistent loss of memory CD4(+)T cells, which are being eliminated by direct virus killing and activation-induced cell death, requires the continuous differentiation of na?ve into memory CD4+ T cells. This unrelenting replenishment process eventually leads to the exhaustion of the na?ve CD4+T-cell pool and the development of disease.     

Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.     

Patterns of CD8+ immunodominance may influence the ability of Mamu-B*08-positive macaques to naturally control simian immunodeficiency virus SIVmac239 replication

Certain major histocompatibility complex (MHC) class I alleles are strongly associated with control of human immunodeficiency virus and simian immunodeficiency virus (SIV). CD8(+) T cells specific for epitopes restricted by these molecules may be particularly effective. Understanding how CD8(+) T cells contribute to control of viral replication should yield important insights for vaccine design. We have recently identified an Indian rhesus macaque MHC class I allele, Mamu-B*08, associated with elite control and low plasma viremia after infection with the pathogenic isolate SIVmac239. Here, we infected four Mamu-B*08-positive macaques with SIVmac239 to investigate why some of these macaques control viral replication. Three of the four macaques controlled SIVmac239 replication with plasma virus concentrations below 20,000 viral RNA copies/ml at 20 weeks postinfection; two of four macaques were elite controllers (ECs). Interestingly, two of the four macaques preserved their CD4(+) memory T lymphocytes during peak viremia, and all four recovered their CD4(+) memory T lymphocytes in the chronic phase of infection. Mamu-B*08-restricted CD8(+) T-cell responses dominated the acute phase and accounted for 23.3% to 59.6% of the total SIV-specific immune responses. Additionally, the ECs mounted strong and broad CD8(+) T-cell responses against several epitopes in Vif and Nef. Mamu-B*08-specific CD8(+) T cells accounted for the majority of mutations in the virus at 18 weeks postinfection. Interestingly, patterns of viral variation in Nef differed between the ECs and the other two macaques. Natural containment of AIDS virus replication in Mamu-B*08-positive macaques may, therefore, be related to a combination of immunodominance and viral escape from CD8(+) T-cell responses.     

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.     

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.     

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.     

Recruitment times, proliferation, and apoptosis rates during the CD8(+) T-cell response to lymphocytic choriomeningitis virus

The specific CD8(+) T-cell response during acute lymphocytic choriomeningitis virus (LCMV) infection of mice is characterized by a rapid proliferation phase, followed by a rapid death phase and long-term memory. In BALB/c mice the immunodominant and subdominant CD8(+) responses are directed against the NP118 and GP283 epitopes. These responses differ mainly in the magnitude of the epitope-specific CD8(+) T-cell expansion. Using mathematical models together with a nonlinear parameter estimation procedure, we estimate the parameters describing the rates of change during the three phases and thereby establish the differences between the responses to the two epitopes. We find that CD8(+) cell proliferation begins 1 to 2 days after infection and occurs at an average rate of 3 day(-1), reaching the maximum population size between days 5 and 6 after immunization. The 10-fold difference in expansion to the NP118 and GP283 epitopes can be accounted for in our model by a 3.5-fold difference in the antigen concentration of these epitopes at which T-cell stimulation is half-maximal. As a consequence of this 3.5-fold difference in the epitope concentration needed for T-cell stimulation, the rates of activation and proliferation of T cells specific for the two epitopes differ during the response and in combination can account for the large difference in the magnitude of the response. After the peak, during the death phase, the population declines at a rate of 0.5 day(-1), i.e., cells have an average life time of 2 days. The model accounts for a memory cell population of 5% of the peak population size by a reversal to memory of 1 to 2% of the activated cells per day during the death phase.     

No one is naive: the significance of heterologous T-cell immunity

Memory T cells that are specific for one virus can become activated during infection with an unrelated heterologous virus, and might have roles in protective immunity and immunopathology. The course of each infection is influenced by the T-cell memory pool that has been laid down by a host's history of previous infections, and with each successive infection, T-cell memory to previously encountered agents is modified. Here, we discuss evidence from studies in mice and humans that shows the importance of this phenomenon in determining the outcome of infection.     

Antiviral CD4 and CD8 T-cell memory: differences in the size of the response and activation requirements

Following acute lymphocytic choriomeningitis virus (LCMV) infection, there is a potent antiviral CD8 T-cell response that eliminates the infection. This initial CD8 T-cell response is followed by a period of memory during which elevated numbers of virus-specific CD8 T cells remain in the mouse. CD4 T cells are also activated after LCMV infection, but relatively less is known about the magnitude and duration of the CD4 response. In this study, we used intracellular staining for interferon-gamma to measure both CD4 and CD8 responses in the same mice at the single cell level. After LCMV infection, there was an increase in the number of activated CD4 T cells and an associated increase in the number of virus-specific CD4 T cells. At the peak of this expansion phase, the frequency of virus-specific CD4 T cells was 1 in 20 (0.5-1.0 x 10(6) per spleen). Like the CD8 response, long-term CD4 memory could be found up to a year after the infection with frequencies of approximately 1 in 260 (0.5-1.5 x 10(5) per spleen). However, the magnitude of virus-specific CD8 T cells was greater than virus-specific CD4 T cells during all phases of the immune response (expansion, death, and memory). At day 8, there were 20- to 35-fold more virus-specific CD8 T cells than CD4 T cells. This initial difference in cell number lasted into the memory phase as there remained a ten- to 20-fold difference in the CD8 and CD4 responses. These results highlight the importance of the expansion phase in determining the size of the memory T-cell pool. In addition to the difference in the magnitude, the activation requirements of CD8 and CD4 T-cell responses were different: CD8 T responses were not affected by blockade of CD40-CD40 ligand interaction whereas CD4 responses were reduced 90%. So while there is long-term memory in both the CD8 and CD4 compartments, the rules regulating the activation of CD8 and CD4 T cells and the overall magnitude of the responses are different.     

Transient antiretroviral treatment during acute simian immunodeficiency virus infection facilitates long-term control of the virus

Experimental evidence and mathematical models indicate that CD4+ T-cell help is required to generate memory cytotoxicT-lymphocyte precursors (CTLp) that are capable of persisting without ongoing antigenic stimulation, and that such responses are necessary to clear an infection or to control it in the long term. Here we analyse mathematical models of simian immunodeficiency virus (SIV) replication in macaques, assuming that SIV impairs specific CD4+ T-cell responses. According to the models, fast viral replication during the initial stages of primary infection can result in failure to generate sufficient long-lived memory CTLp required to control the infection in the long term. Modelling of drug therapy during the acute phase of the infection indicates that transient treatment can minimize the amount of virus-induced immune impairment, allowing a more effective initial immune sensitization. The result is the development of high levels of memory CTLp that are capable of controlling SIV replication in the long term, in the absence of continuous treament. In the model, the success of treatment depends crucially on the timing and duration of antiretroviral therapy. Data on SIV-infected macaques receiving transient drug therapy during acute infection support these theoretical predictions. The data and modelling suggest that among subjects controlling SIV replication most efficiently after treatment, there is a positive correlation between cellular immune responses and virus load in the post-acute stage of infection. Among subjects showing less-efficient virus control, the correlation is negative. We discuss our findings in relation to previously published data on HIV infection.     

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.     

Simian immunodeficiency virus infection induces severe loss of intestinal central memory T cells which impairs CD4+ T-cell restoration during antiretroviral therapy

BACKGROUND: Simian immunodeficiency virus (SIV) infection leads to severe loss of intestinal CD4(+) T cells and, as compared to peripheral blood, restoration of these cells is slow during antiretroviral therapy (ART). Mechanisms for this delay have not been examined in context of which specific CD4(+) memory subsets or lost and fail to regenerate during ART. METHODS: Fifteen rhesus macaques were infected with SIV, five of which received ART (FTC/PMPA) for 30 weeks. Viral loads were measured by real-time PCR. Flow cytometric analysis determined changes in T-cell subsets and their proliferative state. RESULTS: Changes in proliferative CD4(+) memory subsets during infection accelerated their depletion. This reduced the central memory CD4(+) T-cell pool and contributed to slow CD4(+) T-cell restoration during ART. CONCLUSION: There was a lack of restoration of the CD4(+) central memory and effector memory T-cell subsets in gut-associated lymphoid tissue during ART, which may contribute to the altered intestinal T-cell homeostasis in SIV 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.     

Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes

Proinflammatory cytokines secreted by memory CD8+ and CD4+ T cells are thought to play a direct role in the pathogenesis of dengue virus infection by increasing vascular permeability and thereby inducing the pathophysiologic events associated with dengue hemorrhagic fever and dengue shock syndrome. Severe disease is frequently observed in the setting of secondary infection with heterologous dengue virus serotypes, suggesting a role for cross-reactive memory T cells in the immunopathogenesis of severe disease. We used a large panel of well-characterized dengue virus-specific CD8+ T-cell clones isolated from Pacific Islanders previously infected with dengue virus 1 to examine effector memory function, focusing on a novel dominant HLA-B*5502-restricted NS5(329-337) epitope, and assessed T-cell responses to stimulation with variant peptides representing heterologous serotypes. Variant peptides were differentially recognized by dengue virus 1-specific effector CD8+ cytotoxic T lymphocytes (CTL) in a heterogeneous and clone-specific manner, in which cytolytic function and cytokine secretion could be enhanced, diminished, or abrogated compared with cognate peptide stimulation. Dengue virus-specific CTL stimulated with cognate and variant peptides demonstrated a cytokine response hierarchy of gamma IFN (IFN-gamma) > tumor necrosis factor alpha (TNF-alpha) > interleukin-2 (IL-2), and a subset of clones also produced IL-4 and IL-6. Individual clones demonstrated greater avidity for variant peptides representing heterologous serotypes, including serotypes previously encountered by the subject, and IFN-gamma and TNF-alpha secretion was enhanced by stimulation with these heterologous peptides. Altered antiviral T-cell responses in response to stimulation with heterologous dengue virus serotypes have implications for control of virus replication and for disease pathogenesis.     

CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T-cell responses against herpes simplex virus type 1

It has become evident that naturally occurring CD25(+) regulatory T cells (T(reg) cells) not only influence self-antigen specific immune response but also dampen foreign antigen specific immunity. This report extends our previous findings by demonstrating that immunity to certain herpes simplex virus (HSV) vaccines is significantly elevated and more effective if T(reg) cell response is curtailed during either primary or recall immunization. The data presented here show that removal of CD25(+) T(reg) cells prior to SSIEFARL-CpG or gB-DNA immunization significantly enhanced the resultant CD8(+) T-cell response to the immunodominant SSIEFARL peptide. The enhanced CD8(+) T-cell reactivity in T(reg) cell-depleted animals was between two- and threefold and evident in both acute and memory stages. Interestingly, removal of CD25(+) T(reg) cells during the memory recall response to plasmid immunization resulted in a twofold increase in CD8(+) T-cell memory pool. Moreover, in the challenge experiments, memory CD8(+) T cells generated with plasmid DNA in the absence of T(reg) cells cleared the virus more effectively compared with control groups. We conclude that CD25(+) T(reg) cells quantitatively as well as qualitatively affect the memory CD8(+) T-cell response generated by gB-DNA vaccination against HSV. However, it remains to be seen if all types of vaccines against HSV are similarly affected by CD25(+) T(reg) cells and if it is possible to devise means of limiting T(reg) cell activity to enhance vaccine efficacy.     

Immunization with Chaperone-Peptide Complex Induces Low-Avidity Cytotoxic T Lymphocytes Providing Transient Protection against Herpes Simplex Virus Infection

Heat shock proteins loaded with viral peptides were shown to induce a CD8+ T cell response and confer protective immunity against challenge with herpes simplex virus (HSV). The delivery system consisted of recombinant human hsp70 coupled to the peptide SSIEFARL, which is the immunodominant peptide epitope, recognized by HSV specific T cells in C57BL/6 mice. Immunization resulted in CD8+ T-cell responses, measured by peptide-specific tetramers and peptide-induced intracellular gamma interferon expression and cytotoxicity, similar to responses resulting from immunization with a recombinant vaccinia virus that expressed SSIEFARL as a minigene (VvgB) and UV-inactivated HSV. However, the durability of the hsp70-SSIEFARL response was less than that resulting from VvgB and HSV immunization and in addition the CD8+ T-cell responses in the memory phase were functionally less effective. Mice challenged soon after immunization showed excellent immunity, but by 90 days postimmunization this had waned to be significantly less than the level of immunity in both VvgB- and HSV-immunized mice.     

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8⁺ T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8⁺ T cells during the acute and chronic stages of infection. Although HCV-specific CD8⁺ T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8⁺ T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8⁺ T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8⁺ T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.     

Immunological memory and acquired immunodeficiency syndrome pathogenesis

Infection with the human immunodeficiency virus results in profound perturbations in immunological memory, ultimately resulting in increased susceptibility to opportunistic infections and acquired immunodeficiency syndrome (AIDS). We have used rhesus macaques infected with the simian immunodeficiency virus (SIV) as a model to understand better the effects of AIDS virus infection on immunological memory. Acute infection with SIV resulted in significant deficits in CD4+ helper responses to cytomegalovirus (CMV) as well as CMV-specific cytotoxic T-lymphocyte and neutralizing antibody responses. Reactivation of CMV was associated with high levels of SIV replication and suppression of both T-helper and cytotoxic responses to CMV. We have also studied the effects of SIV infection on T-cell turnover in non-human primates. T-cell turnover was evaluated using the nucleoside analogue bromodeoxyuridine (BrdU) in combination with five-colour flow cytometric analysis. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labelling and elimination rates of both CD4+ and CD8+ BrdU-labelled cells were increased by two- to threefold compared with normal controls. Further analysis of immunological memory in non-human primates should offer the opportunity to extend immunological insights from murine models to the pathogenesis and prevention of AIDS.