HCMV-specific CD8 Research Articles

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

Immature dendritic cells (DC) infected with an endotheliotropic (Huv(+)) and leukotropic (Leuk(+)) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4(+) and CD8(+) T cells. Infected DC were co-cultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-gamma production. Efficient stimulation of HCMV-specific CD4(+) and CD8(+) T cell responses was achieved using DC productively infected with Huv(+) Leuk(+) VR1814 strain. On the contrary, a negligible CD8(+) T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4(+) and CD8(+) T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.

Virus‐Specific Cytotoxic T Lymphocytes Differentially Express Cell‐Surface Leukocyte Immunoglobulin‐Like Receptor–1, an Inhibitory Receptor for Class I Major Histocompatibility Complex Molecules

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8+ T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection.

Dendritic-cell infection by human cytomegalovirus is restricted to strains carrying functional UL131–128 genes and mediates efficient viral antigen presentation to CD8+ T cells

Human cytomegalovirus (HCMV) genetic determinants of endothelial-cell tropism and virus transfer to leukocytes (both polymorphonuclear and monocyte) have been recently identified in the UL131-128 genes. Here it is documented that the same genetic determinants of HCMV are responsible for monocyte-derived dendritic-cell (DC) tropism, i.e. all endotheliotropic and leukotropic strains of HCMV are also DC-tropic (or dendrotropic). In fact, all recent clinical HCMV isolates and deletion mutants sparing the UL131-128 locus as well as the endotheliotropic revertants AD169 and Towne were able to productively infect DC following co-culture with infected endothelial cells. On the contrary, the same clinical isolates extensively propagated in human fibroblasts, the UL131-128 deletion mutants and the reference laboratory strains were not. Peak extracellular virus titres in DC were reached 4-7 days post-infection (p.i.). Viral proteins pp65 and p72 were detected 1-3 h p.i., involving the great majority of DC 24 h p.i., while gB was abundantly detected 96 h p.i., when a cytopathic effect first appeared. Infection of DC with cell-free virus released into the medium could only be achieved with HCMV strains extensively adapted to growth in endothelial cells, reaching the peak titres 10 days p.i. DC infected for 24 h with cell-free virus and incubated for 16 h with autologous peripheral blood mononuclear cells were found to act as a potent stimulator of both HCMV-specific CD4+- and CD8+-mediated immune responses, as determined by cytokine flow cytometry. DC incubated with inactivated crude whole viral antigen preparations were only capable of eliciting a significant CD4+-mediated immune response.

Cross-recognition of human alloantigen by cytomegalovirus glycoprotein-specific CD4+ cytotoxic T lymphocytes: implications for graft-versus-host disease

AbstractPresence of the HLA-DR7 allele in patients who receive transplants has been proposed as a risk factor for human cytomegalovirus (HCMV)–associated complications; however, the precise mechanism of this increased risk remains unresolved. Here we show that HLA-DR7–restricted HCMV-specific CD4+ cytotoxic T lymphocytes (CTLs) can display an unusual dual specificity toward a glycoprotein-B (gB) epitope and the alloantigen HLA-DR4. However, no HLA-DR4–specific alloreactivity was observed when the gB-specific CTLs were generated from virus carriers expressing both HLA-DR7 and DR4 alleles. This most likely demonstrates the clonal inactivation of potentially self-reactive T cells in humans. Fine specificity analysis showed that gB-specific CTLs from HLA-DR7+/DR4- individuals displayed a distinct pattern of recognition when compared with CTLs from HLA-DR7+/DR4+ individuals, presumably evading an area of the epitope that mimics a structure presented on HLA-DR4. These data illustrate a possible mechanism for the clinical association between HCMV and graft-versus-host disease.

T Cell-Mediated Control of HCMV Infection in Pediatric Patients Receiving Hematopoietic Stem Cell Transplantation.

We are studying the development of HCMV-specific CD4+ and CD8+ T cell response after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. A new technique was developed to simultaneously detect HCMV-specific CD4+ and CD8+ effector T cells using HCMV-infected autologous dendritic cells as stimulators and intracellular staining of IFN-γ production by T cells. This prospective study is based on monthly determination of both HCMV-specific T cell number and in vitro lymphoproliferative response to crude HCMV antigen. Patients are routinely monitored for HCMV infection/reactivation in blood (by determination of either antigenemia or quantitation of viral DNA) and treated according to a strategy of pre-emptive therapy. So far, of 41 patients receiving HSCT from an HLA-identical related donor (n=18), unrelated donor (n=15) or a T cell-depleted HSCT from a haploidentical relative (n=8), 25 patients have reached day +180, while 16 patients completed a follow-up of 90 days. Among the 28 HCMV-seropositive HSCT recipients, 25 developed HCMV-specific CD4+ and CD8+ T-cell response within the first 60 days after transplantation. In these patients, absolute CD4+ T cell count increased over time, but remained lower than that of healthy controls also at later time points. By contrast, CD8+ T cells reached and maintained absolute levels comparable to those of controls already from day +60. At this time, HCMV-specific CD4+ T cell count was comparable to that of controls, while HCMV-specific CD8+ T cell count was higher than that of controls, with no significant change thereafter. On the other hand, in vitro lymphoproliferative response to HCMV antigen was detectable only in about one half of these patients, even at day +180.HCMV infection was detected in blood of 22 of the 25 patients in whom HCMV-specific T cells were present. It was either self-limiting (n=14) or in 8 patients required shorter ganciclovir course (median 7 days, range 5-14) than in the 3 HCMV seropositive patients who developed HCMV infection in the absence of specific immunity (median 67 days, range 42–82, p<0.001). No patient developed HCMV disease or late viral infections. Conversely, HCMV-specific response was detected in only 3/13 HCMV seronegative recipients (none of whom developing detectable HCMV infection in blood). In these patients, both absolute and HCMV-specific T cell counts were lower than those of both controls and HCMV-seropositive HSCT recipients. Our data suggest that effective HCMV-specific T cell immunity can promptly develop after HSCT (regardless of donor type or T-cell depletion of the graft), particularly in seropositive recipients in whom latent virus may be a major antigenic drive for rapid reconstitution of T cell compartment, especially of CD8+ lymphocytes. On the other hand, transfer of memory T cell immunity from seropositive donors to seronegative recipients does not appear to be always sufficient to permit detection of virus-specific lymphocytes in patient's peripheral blood in the early period after the allograft, possibly also due to the lower chance of in vivo antigen stimulation. The frequent dissociation between IFN-γ production and lymphoproliferative response remains to be explained. Future studies could address modulation of antiviral intervention on the reconstitution of HCMV-specific T cell immune response.

T Cell Recognition of HCMV: Pan-Genome Analysis of Immunogenic Open-Reading Frames.

Human Cytomegalovirus (HCMV) is among the largest and most complex of known viruses with 150–200nm virions enclosing a double stranded 230kb DNA genome capable of coding for >200 proteins. HCMV infection is life-long, and for the vast majority of immune competent individuals clinically benign. Disease occurs almost exclusively in the setting of immune deficiency, suggesting that the stable host-parasite relationship that characterizes these infections is the result of an evolutionarily “negotiated” balance between viral mechanisms of pathogenesis and the host immune response. In keeping with, and perhaps because of this balance, the human CD4+ T cell response to whole HCMV viral lysates is enormous, with median peripheral blood frequencies of HCMV-specific cells ~5–10 fold higher than for analogous preparations of other common viruses. Although certain HCMV ORFs have been identified as targets of either the CD4+ or CD8+ T cell response, the specificities comprising the CD4+ T cell response, and both the total frequencies and component parts of the CD8+ T cell response are unknown. Here, we used cytokine flow cytometry and ~14,000 overlapping 15mer peptides comprising all 213 HCMV ORFs encoding proteins >100 amino acids in length to precisely define the total CD4+ and CD8+ HCMV-specific T cell responses and the HCMV ORFs responsible for these responses in 33 HCMV-seropositive, HLA-disparate donors. An additional 9 HCMV seronegative donors were similarly examined to define the extent to which non-HCMV responses cross-react with HCMV-encoded epitopes. We found that when totaled, the median frequencies of HCMV-specific CD4+ and CD8+ T cells in the peripheral blood of the seropositive subjects were 4.0% and 4.5% for the total CD4+ or CD8+ T cell populations, respectively (which corresponds to 9.1% and 10.5% of the memory populations, respectively). The HCMV-specific CD4+ and CD8+ T cell responses included a median 12 and 7 different ORFs, respectively, and all told, 73 HCMV ORFs were identified as targets for both CD4+ and CD8+ T cells, 26 ORFs as targets for CD8+ T cells alone, and 43 ORFS as targets for CD4+ T cells alone. UL55, UL83, UL86, UL99, and UL122 were the HCMV ORFs most commonly recognized by CD4+ T cells; UL123, UL83, UL48, UL122 and UL28 were the HCMV ORFs most commonly recognized by CD8+ T cells. The relationship between immunogenicity and 1) HLA haplotype and 2) ORF expression and function will be discussed. HCMV-seronegative individuals were non-reactive with the vast majority of HCMV peptides. Only 7 potentially cross-reactive responses were identified (all by CD8+ T cells) to 3 ORFs (US32, US29 and UL116) out of a total of almost 4,000 potential responses, suggesting fortuitous cross-reactivity with HCMV epitopes is uncommon. These data provide the first glimpse of the total human T cell response to a complex infectious agent, and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Phenotypic classification of human CD8+ T cells reflecting their function: inverse correlation between quantitative expression of CD27 and cytotoxic effector function.

Phenotypic classification of human CD8(+) T cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8(+) T cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8(+) T cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8(+) T cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8(+) T cells that more precisely reflects their function, the CD27(+) subset was divided into CD27(low) and CD27(high) subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8(+) T cells. HCMV-specific CD8(+) T cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8(+) T cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8(+) T cells using this classification will enable the characterization of antigen-specific CD8(+) T cells.

Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells.

Dendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early-early kinetics, and viral interference with NF-kappaB signaling is likely to be at least partially responsible for the lack of CCR7 expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they do not stimulate HCMV-specific CD8(+) T cells, while CD4(+)-T-cell activation is not impaired. Although CD8(+) T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8(+)-T-cell responses and aiding early viral spread.

Late diversification in the clonal composition of human cytomegalovirus-specific CD8+ T cells following allogeneic hemopoietic stem cell transplantation

AbstractTo investigate the mechanisms of human T-cell reconstitution following allogeneic hemopoietic stem cell transplantation (alloSCT), we analyzed the clonal composition of human cytomegalovirus (HCMV)-specific or Epstein-Barr virus (EBV)-specific CD8+ T cells in 10 alloSC transplant recipients and their donors. All virus-specific CD8+ T-cell clones isolated from recipients after alloSCT contained DNA of donor origin. In all 6 D+/R+ sibling alloSCTs from seropositive donors into seropositive recipients, donor virus-specific clones transferred in the allograft underwent early expansion and were maintained long term in the recipient. In contrast, in 2 of 3 HCMV D+/R- alloSC transplant recipients in whom there was no detectable HCMV infection, donor HCMV-specific clones were undetectable, whereas donor EBV-specific clones were maintained in the same EBV-seropositive recipients, suggesting that transferred clones require antigen for their maintenance. Following D-/R+ transplantation from 3 seronegative donors into seropositive recipients, a delayed primary virus-specific CD8+ T-cell response was observed, in which the T cells contained donor DNA, suggesting that new antigen-specific T cells arose in the recipient from donor-derived progenitors. In 2 of 4 HCMV D+/R+ sibling allograft recipients the clonal composition underwent diversification as compared with their donors, with delayed persistent expansion of HCMV-specific clones that were undetectable in the donor or in the recipient during the early months after transplantation; this diversification may represent expansion of new clones generated from donor-derived progenitors. We conclude that, following alloSCT, late diversification of the HCMV-specific CD8+ T-cell clonal repertoire can occur in response to persistent viral antigen.

Monitoring of human cytomegalovirus (HCMV)-specific CD4+ T cell frequency by cytokine flow cytometry as a possible indicator for discontinuation of HCMV secondary prophylaxis in HAART-treated AIDS patients.

Absolute CD4+ T cell count and human cytomegalovirus (HCMV)-specific CD4+ T cell frequency (as determined by cytokine flow cytometry, CFC) were compared for their ability to predict HCMV disease and safe discontinuation of HCMV secondary prophylaxis. Three groups of AIDS patients with previous nadir CD4+ T cell count <100/microl were studied. Group A included 48 HAART-treated patients with no HCMV disease. Group B included 11 HAART-treated patients with previous HCMV disease who discontinued HCMV prophylaxis. Group C included 23 HAART-treated (n = 16) or -naive (n = 7) patients with previous HCMV disease either continuing or starting HCMV prophylaxis. Patients underwent follow-up for detection of HCMV viremia or disease (groups A and B) and for discontinuation of HCMV secondary prophylaxis on the basis of either HCMV-specific or absolute CD4+ T cell count (group C). During follow-up, while some patients showed a stable HCMV-specific CD4+ T cell response, others had a fluctuating response (unstable responders) or showed no response at all. In detail, 13/48 group A patients were either HCMV non-responders or unstable responders and 2 of them developed HCMV viremia; 3/11 group B patients were unstable responders, none developing either HCMV viremia or disease; finally, 9 group C patients discontinued HCMV prophylaxis based on absolute CD4+ T cell count > 150 cells/microl, but in 2 of them lacking HCMV-specific response HCMV retinitis relapsed. None of the seven group C patients discontinuing HCMV prophylaxis on the basis of CFC showed HCMV disease relapse. CFC may support absolute CD4+ T cell count for both guiding HCMV prophylaxis discontinuation and better monitoring HCMV infection in AIDS patients with no previous HCMV disease or having discontinued HCMV prophylaxis.

Human cytomegalovirus infection during pregnancy and detection of specific T cells by intracellular cytokine staining

Objective: The flow cytometric assay was evaluated as a tool for real-time monitoring of human cytomegalovirus (HCMV)-specific cellular immunity in pregnant women. Methods: We screened for HCMV infection in pregnant women in Sapporo, Japan, during the year 2000, by serologic assays, virus isolation from urine, and PCR to detect DNA in cervical swabs. The frequencies of HCMV-specific CD4 + T cells in pregnant women with serum anti-HCMV IgG antibody were detected by intracellular cytokine (ICC), interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) staining. Results: The levels of intracellular cytokines in pregnant women with serum anti-HCMV IgG antibody were significantly higher than those in women without anti-HCMV IgG antibody (P = 0.011 for IFN-γ and P = 0.023 for TNF-α) but lower than those in non-pregnant women with serum anti-HCMV IgG antibody. Frequencies of HCMV-specific CD4 + T cells were higher in infants with symptomatic congenital infection than in infants with asymptomatic perinatal infection. Conclusions: This ICC assay may reflect immunologic activity against HCMV infection in pregnant women with immunosuppressive conditions.

Mature CD8+ T lymphocyte response to viral infection during fetal life

Immunization of newborns against viral infections may be hampered by ineffective CD8+ T cell responses. To characterize the function of CD8+ T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8+ T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8+ T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28–CD27+CD45RO+, perforinlow), and they acquired a late differentiation phenotype (CD28–CD27-CD45RA+, perforinhigh) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8+ T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Mature CD8(+) T lymphocyte response to viral infection during fetal life.

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers.

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype.

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.

Sensitive detection of human cytomegalovirus peptide–specific cytotoxic T-lymphocyte responses by interferon-γ–enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation

Reconstitution of human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-gamma release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8(+) T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide-specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen-specific CD4(+) T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35->180 days). Peak counts of peptide-specific CD8(+) T cells ranged from 0.14 to 60.6 cells/microL; those of protein-specific CD4(+) T cells ranged from 0.64 to 18.97 cells/microL. Reconstitution of HCMV-peptide-specific CD8(+) T cells and protein-specific CD4(+) T cells was associated with clearance of HCMV infection (r(2) = 0.89, P <.0001 and r(2) = 0.61, P =.0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein-specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4(+) and CD8(+) T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.

Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression.

Memory T cells specific for varicella-zoster virus (VZV), herpes simplex virus (HSV), and human cytomegalovirus (HCMV) were compared in immune adults by intracellular cytokine (ICC) detection. The mean percentages of CD4+ T cells were 0.11% for VZV and 0.22% for HSV by interferon (IFN)-gamma production; the frequency for HCMV was significantly higher at 1.21%. Percentages of VZV-, HSV-, and HCMV-specific CD4+ T cells were similar by use of tumor necrosis factor (TNF)-alpha. HCMV-stimulated CD8+ T cells produced IFN-gamma (1.11%) and TNF-alpha (1.71%); VZV- and HSV-specific CD8+ T cells were not detectable. VZV CD4+ T cell numbers were similar in young adults with natural or vaccine-induced immunity. VZV CD4+ T cells were significantly less frequent in older adults. Secondary varicella immunization did not increase VZV-specific CD4+ T cell frequencies by ICC assay. Numbers of memory T cells specific for herpesviruses may vary with sites of viral latency and with host age.

Expression of Human Cytomegalovirus Immediate-Early Protein IE1 in Insect Cells: splicing of RNA and Recognition by CD4+ T-Cell Clones

Recombinant baculoviruses containing the unspliced gene (Bac-IE1) and a truncated cDNA (Bac-EX4) of the immediate early protein 1 (IE1) of human cytomegalovirus (HCMV) were constructed. The recombinant proteins IE1 and EX4 were expressed in Sf 9 insect cells. Immunoblot analyses using a specific monoclonal antibody or human sera from HCMV seropositive subjects revealed that the IE1 protein had an apparent molecular mass of 71 kDa which was similar to that observed in both HCMV infected human fibroblasts and infected or transfected human astrocytoma cells. Furthermore, HCMV-specific CD4+ T cell clones proliferated in the presence of IE1 or of EX4 used as a control, and appropriate antigen presenting cells. Our data on the IE1 gene provide evidence that two introns can be properly spliced out in baculovirus infected insect cells. The expressed proteins should be useful in further studies on the immune response to the virus.