Lungs are a major organ site of cytomegalovirus latency and recurrence

Recurrence of infectious virus from the latent viral genomes is the initiating event in the pathogenesis of cytomegalovirus (CMV) disease during states of immunodeficiency. Interstitial pneumonia is a frequent manifestation of posttransplantation CMV disease, in particular after bone marrow transplantation and heart and lung transplantations. Recurrence can occur within the transplant derived from a latent infected donor as well as within latently infected organs of the transplant recipient. The reason for a predilection of the lungs as a site of CMV pathology is so far unknown. In a murine model of CMV latency, the lungs were identified as an authentic site of latent infection, since the viral genome remained detectable in lung tissue even after it was cleared to an undetectable level in blood and bone marrow. A comparison between the lungs and the spleen, the previously most thoroughly investigated site of murine CMV latency, revealed a 10-fold-higher burden of latent viral genome for the lungs. Most important, the organ-specific risk of in vivo recurrence was found to correlate with the organ-specific viral genomic load. This new finding thus characterizes the lungs as a high-risk organ for CMV recurrence, and this fact may explain in part why interstitial pneumonia is a frequent manifestation of recurrent CMV infection.     

Murine cytomegalovirus DNA in peripheral blood of latently infected mice is detectable only in monocytes and polymorphonuclear leukocytes

Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.     

Control of cytomegalovirus in bone marrow transplantation chimeras lacking the prevailing antigen-presenting molecule in recipient tissues rests primarily on recipient-derived CD8 T cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule Ld were transplanted into the Ld gene deletion mutant BALB/c-H-2(dm2), an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of Ld-positive donor-derived and Ld-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against Ld. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by Ld within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.     

Clinical utility of tetramer-based immune monitoring in allogeneic stem cell transplantation

The construction and use of Class I human leucocyte antigen (HLA) tetramers has, for the first time, allowed the direct enumeration of CD8(+) T lymphocytes specific for the antigen of interest. Tetramer staining can be combined with functional assays of antigen-specific T cells measuring their production of intracellular cytokines after short-term stimulation with antigen. The advantages of flow cytometric tetramer-based assays are their short turn-around time and their amenability to standardisation. Currently, their main limitation is that only a limited number of Class I HLA tetramers are available. This situation may bias the information derived from such studies. Nevertheless, clinically useful information has been obtained in tetramer-based studies of the regeneration of cytomegalovirus (CMV) and Epstein-Barr virus (EBV)-specific CD8(+) T cells after allogeneic stem cell transplantation (SCT). Following myeloablative cytoreductive therapy and SCT, a period of deep cellular immunodeficiency follows until the donor-derived immune system has sufficiently regenerated. As a result of the lack of immunosurveillance, endogenous viruses such as CMV and EBV may reactivate and cause disease. In order to prevent these complications, pre-emptive therapeutic interventions are made, in which antiviral treatment is administered based on frequent viral load measurements. In this way, overtreatment associated with prophylactic strategies is reduced; however, a subgroup of patients developing recurrent reactivations and/or disease remains. Interventions aimed at the prevention of graft versus host disease (GVHD) and the reduction of its severity greatly reduce the rate of (virus-specific) T-cell reconstitution and hence, increase the frequency and severity of viral reactivations. Specifically, T-cell depletion of SCT and the use of antithymocyte globulin as part of the conditioning regimen reduces the (virus-specific) T lymphocytes transferred with the graft, which otherwise would have contributed to the first phase of T-cell regeneration post-SCT. Consequently, patients whose CMV- and EBV-specific CD8(+) T cells fail to regenerate to levels detectable by tetramer-based flow cytometry during the first 3-6 months post-SCT were at highly increased risk for CMV disease and EBV(+) B-lymphoproliferative disease, respectively. Furthermore, delayed reconstitution of the CD4(+) T-cell compartment results in the lack of adequate help for the generation of sufficient antiviral CD8(+) T cells. Conversely, adoptive immunotherapy using CMV- or EBV-specific T cells results in the swift restoration of virus-specific T-cell immunity and the reduction of viral load, unless corticosteroids have to be administered to treat concurrent GVHD. Studies have shown the clinical utility of tetramer-based immune monitoring in the setting of allogeneic SCT and indicate that such assays, extended by functional studies of the virus-specific T cells, may constitute a valuable extension of current viral load monitoring strategies.     

Do viral load and CD8 cell count at initiation of tritherapy influence the increase of CD4 T-cell count?

BACKGROUND: Tritherapies including protease inhibitors improve clinical status and usually increase CD4 T cell count. However, the dissociation between the marked decreases in viral load and the incomplete restoration of CD4 cell counts with a three-drug combination has been reported. We assessed this potential difference among our patients. METHODS: Patients were enrolled when a protease inhibitor was prescribed to them for the first time. Using a computerized medical record (ADDIS), we retrospectively assessed a potential relationship between the increase in CD4 T cells (deltaCD4) at M3, M6 and variables including sex, age, CDC staging, protease inhibitor, prior antiviral therapy, CD8 and viral load at baseline. We used Epi-Info 6.4 and BMDP software. RESULTS: Data were analyzed on 154 patients. The median CD4 T cell count was 157 at baseline, 215 at month 3 and 202 at month 6. The median viral load was 52000 copies at baseline, 530 at month 3 and 500 at month 6. In a univariate analysis, a significant relationship was found between deltaCD4 and CD8 at baseline. A statistically significant negative correlation appeared between the CD8 cell count at baseline and deltaCD4 at M6 (r=-0.28, Pearson). Moreover, we found that there also was a relationship between deltaCD4 and viral load at baseline. There was a correlation between deltaCD4 at M6 and the viral load at M0 (r=0.37, Pearson). In a multiple regression model, after CD8 count at baseline had been accounted for, we found a significant correlation between deltaCD4 and viral load at baseline (multiple r=0.33 at M3, and 0.40 at M6). CONCLUSIONS: Patients with a low viral load do not benefit from as great an increase in CD4 T cell count as others when they receive a tritherapy including protease inhibitors. These results suggest that another mechanism rather than direct viral pathogenicity leads to CD4 T cell destruction. This mechanism may not be efficiently stopped by antiviral therapy, especially protease inhibitors.     

Diagnostic value of Western blotting in carbohydrate-deficient glycoprotein syndrome

Cytomegalovirus (CMV) infection has been associated with graft rejection in solid organ transplantation and with graft-versus-host disease in marrow transplantation. We hypothesized that CMV-infected endothelial cells play an important role in the rejection process, because of their strategic localization at the interface with the host immune system and their ability to modulate T cell function. To study the effect of CMV infection on cell-mediated cytotoxicity against endothelial cells, peripheral blood mononuclear cells (MNC) were incubated with CMV-infected umbilical vein endothelial cells (CMV-UVEC) or mock-infected controls (M-UVEC) and lysis measured by [3H]leucine release. MNC lysed only CMV-UVEC to a maximum of 23% at E:T 20:1. Lysis was not affected by CD3+ cell depletion, but was abolished by CD16+ cell depletion, indicating that NK cells were the effectors. The kinetics of the NK-mediated lysis of CMV-UVEC paralleled the time course of CMV antigen expression. Furthermore, ganciclovir treatment of CMV-UVEC cultures decreased both specific antigen synthesis and NK-mediated lysis. This indicated that NK might recognize either a viral antigen or a cellular antigen modulated by CMV infection. Treatment of CMV-UVEC with F(ab)2 fragments of human polyclonal anti-CMV antibodies failed to inhibit NK cytotoxicity. In contrast, F(ab)2 fragments of MB40.5, a murine MAb reactive with a conserved epitope on the human MHC class I, significantly decreased lysis, proving that NK lysis of CMV-UVEC is an MHC class I-dependent function. To determine whether CMV-UVEC lysis was dependent solely on upregulation of MHC class I, MNC were incubated with CMV-UVEC mixed with uninfected UVEC. There was no competition for NK-target recognition sites, indicating that NK lysis required an interaction with an MHC class I antigen modified by viral infection. Antibodies against IFN-alpha or -beta did not block NK cytotoxicity against CMV-UVEC. Our findings provide a working frame for further evaluation of cellular immune responses to CMV infection.     

Contributions of antibody and T cell subsets to protection elicited by immunization with a replication-defective mutant of herpes simplex virus type 1

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus, d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depleted in vivo of CD4+ or CD8+ T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+ T cells but not by depletion of CD8+ T cells keratitis developed only in a portion of the CD8+ T cell-depleted mice, suggesting that an immunopathologic potential of CD4+ T cells is held in check when immune CD8+ T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+ T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+ T cells in regulating the immunopathologic activity of CD4+ T cells.     

Utility of major leukocyte subpopulations for monitoring secondary cytomegalovirus infections in renal-allograft recipients by PCR

The feasibility of the major peripheral blood leukocyte (PBL) subsets for use in qualitative and quantitative PCR to monitor secondary cytomegalovirus (CMV) infection and ganciclovir therapy was assessed with 188 blood samples derived from 40 CMV immunoglobulin G-positive renal-allograft recipients. In pp65 antigen-positive patients all leukocyte fractions, but only 79.5% of plasma preparations, were PCR positive. In pp65 antigen-negative samples from patients after antiviral treatment only 7.3% of polymorphonuclear cell (PMNL) samples, but 81.8% of peripheral blood mononuclear cells (PBMC), and 10.9% of plasma samples remained PCR positive. Similarly, in patients with latent infections only 5.0% of PMNL, but 51.7% of PBMC preparations, and 8.0% of plasma samples were PCR positive. Regarding patients with active CMV infection, CMV DNA copy numbers in PMNL correlated significantly with pp65 antigen-positive cell counts before and after onset of ganciclovir therapy. Significant differences in CMV DNA copy numbers in PMNL and plasma were observed (i) between patients with symptomatic infection and those with asymptomatic infection and (ii) between patients with active infection and those with latent infection. In contrast, PBMC harbored equally low CMV DNA levels both in patients with active infection and those with latent infections, and no decline of CMV DNA load in PBMC was observed during antiviral treatment. We conclude that detection of CMV DNA in PMNL, not in PBMC, is associated with active infections and is more sensitive than detection of CMV DNA in plasma. Negative PCR results for PMNL after antiviral therapy indicate recovery, and fewer unwanted positive results occur compared to PBMC and plasma. Therefore, purified PMNL should be preferred for analysis by qualitative CMV PCR to avoid unwanted positive results. The CMV DNA load in PBMC compared with that in PMNL is negligible during active infection, so mixed PBL are sufficient for use in quantitative PCR.     

A homologue of the 19 kDa signal recognition particle protein locus in Drosophila melanogaster

AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body.     

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.     

Patterns of expenditures and use of services among older adults with diabetes. Implications for the transition to capitated managed care

BACKGROUND: Rapid quantifiable diagnostic techniques for the diagnosis of cytomegalovirus (CMV) infection may predict patients at risk of CMV pneumonitis and allow preemptive antiviral treatment. METHODS: Using CMV antigenemia as a prospective surveillance technique for CMV infection, we compared the outcome of preemptive treatment (PT) with ganciclovir, 10 mg/kg/day for 21 days directed by "high levels" of CMV antigenemia (PT group, n= 19), with the outcome in a group of historical controls (n=18) treated with ganciclovir when CMV illness occurred. Greater than 50 antigen-positive cells per 2 x 10(5) polymorphonuclear leukocytes was considered to be high-level antigenemia. RESULTS: Nine of the 18 controls developed high-level CMV antigenemia at a median of 33 days (range: 13-65 days) and 5 of the 9 developed CMV disease. Ten of the 19 PT group had high levels of CMV antigenemia detected at a median of 47 days (range: 20-63 days) and were given ganciclovir; none developed CMV disease. There was a significantly lower incidence of CMV disease in the PT group in comparison to controls (0 of 19 vs. 5 of 18: P=0.019). CONCLUSION: We have reduced the incidence of CMV disease using preemptive treatment, and because of a 100% negative predictive value, we omitted unnecessary antiviral prophylaxis for many at-risk patients.     

Expression of polysialylated NCAM but not L1 or N-cadherin by regenerating adult mouse optic fibers in vitro

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.     

Long-term predictive value of a single cytomegalovirus (CMV) DNA PCR assay for CMV disease in human immunodeficiency virus-infected patients

Universal prophylaxis with oral ganciclovir is not cost-effective for the prevention of cytomegalovirus (CMV) disease in human immunodeficiency virus infection. For a preemptive strategy to be considered, patients at highest risk for CMV disease need to be easily and accurately identified. In this study, the sensitivity, specificity, positive predictive value, and negative predictive value of a single CMV DNA PCR assay for the subsequent development of CMV disease were 0.75, 0.89, 0.75, and 0.89, respectively.     

Lack of coordinate control of ferritin and transferrin receptor expression during rat liver regeneration

Cytomegalovirus (CMV) is the most important infectious agent in transplant recipients. The critical step in the pathogenesis of CMV infection is the reactivation of latent virus, which is affected by the immunosuppressive therapy and/or alloantigenic stimulation. The clinical effects of CMV infection include CMV disease (syndrome), an immunosuppressed state, and allograft injury. Recently, the incidence of serious CMV diseases after transplant has been decreased, probably due to the advance in the method for rapid diagnosis, the ganciclovir administration, and the effective prevention of CMV diseases. Seronegative or filtered blood products, CMV immune globulin, and prophylactic or preemptive therapy with ganciclovir appear contribute to the improvement in the prophylaxis for CMV diseases after transplant. Antigenemia-guided early treatment may be promising for the effective prevention of CMV diseases after transplant.     

A role for perforin in downregulating T-cell responses during chronic viral infection

Cytotoxic T cells secrete perforin to kill virus-infected cells. In this study we show that perforin also plays a role in immune regulation. Perforin-deficient (perf -/-) mice chronically infected with lymphocytic choriomeningitis virus (LCMV) contained greater numbers of antiviral T cells compared to persistently infected +/+ mice. The enhanced expansion was seen in both CD4 and CD8 T cells, but the most striking difference was in the numbers of LCMV-specific CD8 T cells present in infected perf -/- mice. Persistent LCMV infection of +/+ mice results in both deletion and anergy of antigen-specific CD8 T cells, and our results show that this peripheral "exhaustion" of activated CD8 T cells occurred less efficiently in perf -/- mice. This excessive accumulation of activated CD8 T cells resulted in immune-mediated damage in persistently infected perf -/- mice; approximately 50% of these mice died within 2 to 4 weeks, and mortality was fully reversed by in vivo depletion of CD8 T cells. This finding highlights an interesting dichotomy between the role of perforin in viral clearance and immunopathology; perforin-deficient CD8 T cells were unable to clear the LCMV infection but were capable of causing immune-mediated damage. Finally, this study shows that perforin also plays a role in regulating T-cell-mediated autoimmunity. Mice that were deficient in both perforin and Fas exhibited a striking acceleration of the spontaneous lymphoproliferative disease seen in Fas-deficient (lpr) mice. Taken together, these results show that the perforin-mediated pathway is involved in downregulating T-cell responses during chronic viral infection and autoimmunity and that perforin and Fas act independently as negative regulators of activated T cells.     

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.     

Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.