High-level primary CD8(+) T-cell response to human immunodeficiency virus type 1 gag and env generated by vaccination with recombinant vesicular stomatitis viruses

We investigated the primary cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (rVSVs). The primary response to Env peaked 5 to 7 days after intraperitoneal vaccination, at which time 40% of CD8(+) cells were Env tetramer positive and activated (CD62L(Lo)). These freshly isolated cells actively lysed target cells pulsed with the p18-I10 peptide and secreted gamma interferon and tumor necrosis factor alpha after stimulation with the Env p18-I10 peptide. The primary response to Env elicited by rVSVs was sixfold higher than that elicited by recombinant vaccinia viruses (rVVs) at 5 days postvaccination. An intranasal route of vaccination with VSV-Env also elicited a strong primary response to Env. The primary immune response to Gag elicited by rVSV peaked 7 days after vaccination, at which time 3% of CD8(+) cells were Gag tetramer positive and CD62L(Lo) and functional by intracellular cytokine staining. This response was eightfold higher than that elicited by rVV expressing Gag. VSV-GagEnv, which expresses both Gag and Env from a single recombinant, also induced strong cytotoxic T-lymphocyte (CTL) responses to both Env and Gag. Our quantitative analyses illustrate the potency of the VSV vector system in CTL induction.     

The virus-specific and allospecific cytotoxic T-lymphocyte response to lymphocytic choriomeningitis virus is modified in a subpopulation of CD8(+) T cells coexpressing the inhibitory major histocompatibility complex class I receptor Ly49G2

The role of negatively signaling NK cell receptors of the Ly49 family on the specificity of the acute CD8(+) cytotoxic T-lymphocyte (CTL) response was investigated in lymphocytic choriomeningitis virus (LCMV)-infected C57BL/6 mice. Activated CD8(+) T cells coexpressing Ly49G2 expanded during LCMV infection, and T-cell receptor analyses by flow cytometry and CDR3 spectratyping revealed a unique polyclonal T-cell population in the Ly49G2(+) fraction. These cells lysed syngeneic targets infected with LCMV or coated with two of three LCMV immunodominant peptides examined. Transfection of these sensitive targets with H2D(d), a ligand for Ly49G2, inhibited lysis. This was reversed by antibody to Ly49G2, indicating effective negative signaling. LCMV characteristically induces an anti-H2(d) allospecific T-cell response that includes T-cell clones cross-reactive between allogeneic and LCMV-infected syngeneic targets. The CD8(+) Ly49G2(+) population mediated no allospecific killing, nor was any NK-like killing observed against YAC-1 cells. This study shows that CD8(+) Ly49G2(+) cells participate in the virus-induced CTL response but lyse a more restricted range of targets than the rest of the virus-induced CTL population.     

Role of CD8(+)T cells in the host response to Chlamydia

Chlamydia infections constitute a major public health problem. Although multiple arms of the immune system participate in the control of Chlamydia in infected hosts, T lymphocytes are essential. This review focuses on the roles that CD8(+)T cells may play in immunoprotection and immunopathology following recognition of Chlamydia-infected cells.     

An early, abundant cytotoxic T-lymphocyte response against Theiler's virus is critical for preventing viral persistence.

In genetically susceptible strains of mice, the DA strain of Theiler's virus, a picornavirus, causes a persistent infection of the white matter of the spinal cord associated with chronic demyelination. In resistant strains, on the other hand, the infection is cleared within 1 to 2 weeks. In this article, we show that Theiler's virus induces a rapid and abundant cytotoxic T lymphocyte (CTL) response in resistant C57BL/6 mice, while the response remains low throughout infection in susceptible SJL/J mice. This difference can be referred to a higher number of virus-specific CTL precursors in C57BL/6 mice. These observations indicate that the efficient induction of virus-specific CTL precursors is critical for avoiding the establishment of a persistent picornaviral infection.     

B7/CD28 costimulation is critical in susceptibility to Pseudomonas aeruginosa corneal infection: a comparative study using monoclonal antibody blockade and CD28-deficient mice

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.     

The membrane-proximal region of vesicular stomatitis virus glycoprotein G ectodomain is critical for fusion and virus infectivity

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.     

Differential dynamics of CD4(+) and CD8(+) T-lymphocyte proliferation and activation in acute simian immunodeficiency virus infection

Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67(+) lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67(+) CD8(+) T lymphocytes and a 2- to 3-fold increase in Ki-67(+) CD3(-) CD8(+) natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67(+) CD4(+) T lymphocytes during acute infection, although transient increases in Ki-67(-) and Ki-67(+) CD4(+) T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4(+) T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67(+) CD8(+) T lymphocytes were phenotypically CD45RA(-) CD49d(hi) Fas(hi) CD25(-) CD69(-) CD28(-) HLA-DR(-) and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8(+) T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4(+) and CD8(+) T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

The cytotoxic T-lymphocyte response to Sendai virus is unimpaired in the absence of gamma interferon.

Sendai virus is eliminated from the respiratory tract of gamma interferon (IFN-gamma) -/- BALB/c mice with normal kinetics. The level of virus-specific cytotoxic T-lymphocyte (CTL) activity in the cell population recovered by bronchoalveolar lavage is unimpaired, the prevalence of interleukin-4 (IL-4)-producing cells is increased, and the titers of virus-specific immunoglobulins IgG1 and IgG2b are higher in the IFN-gamma -/- mice. The emergence of this T-helper 2 response profile in both lymphoid tissue and the pneumonic lung has no obvious deleterious consequences. Virus clearance is slightly delayed following depletion of the CD4+ subset, with the effect being similar in magnitude for IFN-gamma -/- and +/+ mice. However, the generation of CTL precursors (CTLp) is diminished in the IFN-gamma -/- (but not +/+) mice in the absence of concurrent CD4+ T help. Apparently the clonal expansion of the CTLp population can be promoted either by a cytokine (perhaps IL-2) produced by the IFN-gamma -/- CD4+ T cells or by IFN-gamma made by other cell types in the +/+ mice.     

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.     

Type 2 cytokines predominate in the human CD4(+) T-lymphocyte response to Epstein-Barr virus nuclear antigen 1

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persistently infects 85% of the adult population worldwide. In this report, we examine the proliferative response and cytokine secretion profile of CD4(+) T lymphocytes from a panel of unrelated EBV-positive donors against two EBV latent antigens, EBNA1 and EBNA3C. Substantial proliferative responses by CD4(+) lymphocytes were demonstrated to both antigens in multiple, randomly selected donors. Surprisingly, we observed a striking and consistent difference in cytokine response to EBNA1 and EBNA3C. EBNA1-specific CD4(+) T lymphocytes from multiple unrelated donors preferentially produced type 2-like cytokines in response to antigenic stimulation, while the response to EBNA3C was a characteristic type 1 response. The implications of these findings for EBV persistence and the development of EBV-associated malignancies are discussed.     

Dendritic cell-derived IL-12 is not required for the generation of cytotoxic, IFN-gamma-secreting, CD8(+) CTL in vivo.

By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.     

Direct CD28 costimulation is required for CD8+ T cell-mediated resistance to an acute viral disease in a natural host

Previous studies have suggested that, differing from model Ags, viruses that replicate extensively in the host still induce normal CD8+ T cell responses in the absence of CD28 costimulation. Because these studies were performed with viruses that do not normally cause acute disease, an important remaining question is whether CD28 costimulation is required for CD8+ T cell-mediated resistance to widely replicating but pathogenic viruses. To address this question, we studied the role of CD28 costimulation in CD8+ T cell-mediated resistance to mousepox, a disease of the mouse caused by the natural mouse pathogen, the ectromelia virus (ECTV). C57BL/6 (B6) mice are naturally resistant to mousepox, partly due to a fast and strong CD8+ T cell response. We found that B6 mice deficient in CD28 (CD28 knockout (KO)) are highly susceptible to lethal mousepox during the early stages of ECTV infection but can be protected by immunization with the antigenically related vaccinia virus (VACV) or by adoptive transfer of CD28 KO anti-VACV memory CD8+ cells. Of interest, a thorough comparison of the CD8+ T cell responses to ECTV and VACV suggests that the main reason for the susceptibility of CD28 KO mice to mousepox is a reduced response at the early stages of infection. Thus, while in the absence of CD28 costimulation the end point strength of the T cell responses to nonpathogenic viruses may appear normal, CD28 costimulation increases the speed of the T cell response and is essential for resistance to a life-threatening acute viral disease.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

Antigen-presenting B cells: efficient uptake and presentation by activated B cells for induction of cytotoxic T lymphocytes against vesicular stomatitis virus

We have evaluated the efficacy of mitogen (LPS/DxSO4)-activated B cells (B lymphoblasts) to function as antigen-presenting cells (APC) for vesicular stomatitis virus (VSV). Our studies revealed that B lymphoblasts induced potent cytotoxic thymus (T)-derived lymphocyte (CTL) activity in VSV-immune splenic T cells depleted of adherent accessory cells. Dose-response curves indicated that B lymphoblasts were approximately 15-20 times more efficient APC than spleen cells for CTL induction against VSV. There was little evidence of reprocessing of viral antigens by the responder population because only CTL activity restricted to the parental haplotype of the B lymphoblast was generated following stimulation of VSV-immune F1 T cells. B lymphoblasts activated VSV-specific memory CTL which expressed the Lyt-1-23+, AsGM1+ phenotype without activating natural killer and/or lymphokine-activated killer cells. The ability of B lymphoblasts to function as efficient APC was not related to enhanced viral replication in these cells because potent VSV-specific proliferative and class I-restricted CTL responses were induced by B lymphoblasts infected with VSV rendered noninfectious by exposure to ultraviolet (uv) light. This indicates that activated B cells can efficiently process and present input virion protein. Purified splenic B cells that were not activated by mitogen stimulation did not function as APC for VSV even at high multiplicities of infection. The failure of B cells to function as APC for VSV was related to inefficient uptake of VSV and their inability to provide accessory cell signals required for T-cell proliferation; both these functions developed following mitogen stimulation. These data suggest that activated B cells may function as a potent APC population for virus independent of the specificity of their immunoglobulin antigen receptor.     

A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5

Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo.     

I-kappa B kinase beta is critical for B cell proliferation and antibody response.

The NF-kappaB proteins are critical in the regulation of the immune and inflammatory response. Stimulation of the NF-kappaB pathway leads to increases in I-kappaB kinase beta (IKKbeta) kinase activity to result in the enhanced phosphorylation and degradation of I-kappaB and the translocation of the NF-kappaB proteins from the cytoplasm to the nucleus. In this study, a dominant-negative IKKbeta mutant expressed from the IgH promoter was used to generate transgenic mice to address the role of IKKbeta on B cell function. Although these transgenic mice were defective in activating the NF-kappaB pathway in B cells, they exhibited no defects in B lymphocyte development or basal Ig levels. However, they exhibited defects in the cell cycle progression and proliferation of B cells in response to treatment with LPS, anti-CD40, and anti-IgM. Furthermore, selective defects in the production of specific Ig subclasses in response to both T-dependent and T-independent Ags were noted. These results suggest that IKKbeta is critical for the proliferation of B cells and the control of some aspects of the humoral response.     

A critical role for B7/CD28 costimulation in experimental autoimmune encephalomyelitis: a comparative study using costimulatory molecule-deficient mice and monoclonal antibody blockade

The B7/CD28 pathway provides critical costimulatory signals required for complete T cell activation and has served as a potential target for immunotherapeutic strategies designed to regulate autoimmune diseases. This study was designed to examine the roles of CD28 and its individual ligands, B7-1 and B7-2, in experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the CNS. EAE induction in CD28- or B7-deficient nonobese diabetic (NOD) mice was compared with the effects of B7/CD28 blockade using Abs in wild-type NOD mice. Disease severity was significantly reduced in CD28-deficient as well as anti-B7-1/B7-2-treated NOD mice. B7-2 appeared to play the more dominant role as there was a moderate decrease in disease incidence and severity in B7-2-deficient animals. EAE resistance was not due to the lack of effective priming of the myelin peptide-specific T cells in vivo. T cells isolated from CD28-deficient animals produced equivalent amounts of IFN-gamma and TNF-alpha in response to the immunogen, proteolipid protein 56-70. In fact, IFN-gamma and TNF-alpha production by Ag-specific T cells was enhanced in both the B7-1 and B7-2-deficient NOD mice. In contrast, peptide-specific delayed-type hypersensitivity responses in these animals were significantly decreased, suggesting a critical role for CD28 costimulation in in vivo trafficking and systemic immunity. Collectively, these results support a critical role for CD28 costimulation in EAE induction.     

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.