A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.

Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Short-cycle structured intermittent treatment of chronic HIV infection with highly active antiretroviral therapy: effects on virologic, immunologic, and toxicity parameters.

Although continuous highly active antiretroviral therapy (HAART) is effective for many HIV-infected patients, it can be toxic and prohibitive in cost. By decreasing the total amount of time patients receive medications, intermittent HAART could reduce toxicity and cost. Therefore, we initiated a pilot study in which 10 HIV-infected individuals receiving effective therapy that resulted in levels of HIV RNA <50 copies per ml of plasma and CD4(+) T cell counts >300 cells per mm(3) of whole blood received repeated cycles of 7 days on HAART followed by 7 days off of HAART. Patients maintained suppression of plasma viremia for 32-68 weeks. There was no significant increase in HIV proviral DNA or replication-competent HIV in peripheral CD4(+) T cells or HIV RNA in peripheral blood or lymph node mononuclear cells. There was no significant change in CD4(+) T cell counts, no significant increase in CD4(+) or CD8(+) T cells expressing activation markers or producing IFN-gamma in response to HIV, no increase in CD4(+) T cell proliferation to p24 antigen, and no evidence for the development of resistance to HAART medications. There was a significant decrease in serum cholesterol and triglyceride levels. Thus, in this proof-of-concept study, short-cycle intermittent HAART maintained suppression of plasma viremia as well as HIV replication in reservoir sites while preserving CD4(+) T cell counts. In addition, there was a decrease in serum cholesterol and triglyceride levels. Intermittent therapy may be an important strategy to reduce cost and toxicity for HIV-infected individuals.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

CD4 T cell expansions are associated with increased apoptosis rates of T lymphocytes during IL-2 cycles in HIV infected patients.

OBJECTIVE AND DESIGN: In an attempt to determine the mechanisms underlying the CD4 T cell expansions in patients receiving intermittent interleukin (IL)-2, a cohort of 10 HIV infected patients were studied during a 5-day cycle of IL-2 to measure rates of apoptosis, the expression of activation markers in CD4 and CD8 T cell subsets and the serum levels of proinflammatory cytokines. All patients were receiving highly active antiretroviral therapy. METHODS: Peripheral blood mononuclear cells were tested pre- and at the completion of IL-2 treatment with annexin V/7-AAD for the measurement of apoptosis. Phenotypic analyses of T lymphocytes were performed in parallel. Serum levels of interferon (IFN)gamma, granulocyte-macrophage colony stimulating factor, IL-6 and tumor necrosis factor (TNF)alpha were tested by enzyme-linked immunosorbent assay. RESULTS: IL-2 increased the spontaneous apoptosis rates of CD4 and CD8 T lymphocytes (P = 0.003). Expression of HLA-DR, CD38 and CD95 increased on both CD4 and CD8 T lymphocytes whereas CD25 induction was observed exclusively on CD4 T cells. Significant increases of serum IL-6 and TNFalpha levels were noted in all patients whereas viral loads remained unchanged. CONCLUSION: Administration of IL-2 for 5 days in HIV infected patients leads to enhanced apoptosis of both CD4 and CD8 T cells despite an eventual increase of the CD4 T cell count. A profound activation state with induction of activation markers on T cells and high levels of TNFalpha and IL-6 accompanies the increased apoptosis during the IL-2 cycle. These data suggest that the CD4 expansions seen in the context of intermittent IL-2 therapy are the net result of increases in both cell proliferation and cell death.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals.

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21(high)- and CD21(low)-expressing B cells, the CD21(low) fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21(high) fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21(low) B cell population but not in the CD21(high) fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

Interleukin-2 induced immune effects in human immunodeficiency virus-infected patients receiving intermittent interleukin-2 immunotherapy.

To characterize the immunological effects of intermittent IL-2 therapy, which leads to selective increases in CD4+ T lymphocytes in HIV-infected patients, 11 patients underwent extensive immunological evaluation. While IL-2 induced changes in both CD4+ and CD8+ cell number acutely, only CD4+ cells showed sustained increases following discontinuation of IL-2. Transient increases in expression of the activation markers CD38 and HLA-DR were seen on both CD4+ and CD8+ cells, but CD25 (a chain of the IL-2 receptor) increased exclusively on CD4+ cells. This increase in CD25 expression was sustained for months following discontinuation of IL-2, and was seen in naive as well as memory cells. IL-2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL-2 administration. It thus appears that sustained CD25 expression selectively on CD4+ cells is a critical component of the immunological response to IL-2, and that intermittent administration of IL-2 is necessary to overcome the tachyphylaxis to IL-2-induced proliferation.

Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity.

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production.

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.

Longitudinal changes in CD4+ T cell antigen receptor diversity and naive/memory cell phenotype during 9 to 26 months of antiretroviral therapy of HIV-infected patients.

Although skewing of the CD4+ TCR repertoire in advanced HIV infection is well documented, increases in polyclonality during antiretroviral therapy have been less consistently observed. Ten patients, each with documented abnormalities within the CD4+ TCR repertoire, were studied by CDR3 spectratyping, semiquantitative PCR, and SSCP during 9-26 months of therapy. Naive and memory cell phenotypes were analyzed by flow cytometry. Six of 10 patients showed increased polyclonality of their TCR repertoires, 1 showed no change, and 3 showed increased TCR skewing, despite suppressed viral replication. Overall, there was no significant change in the percentage of abnormal BV subfamilies (from a mean of 25.5 to 17.1%) or the percentage of naive CD4+ T cells (from a mean of 18 to 25%). Further, progression of TCR repertoire disruptions was observed in some patients even with suppression of plasma viral RNA below 500 copies/ml. Although a spectrum of changes may be seen within the CD4+ TCR repertoire in the setting of antiretroviral therapy, increases in polyclonality are observed in some patients.

Impact of HIV-1 infection and highly active antiretroviral therapy on the kinetics of CD4+ and CD8+ T cell turnover in HIV-infected patients.

To evaluate the effects of HIV infection on T cell turnover, we examined levels of DNA synthesis in lymph node and peripheral blood mononuclear cell subsets by using ex vivo labeling with BrdUrd. Compared with healthy controls (n = 67), HIV-infected patients (n = 57) had significant increases in the number and fraction of dividing CD4(+) and CD8(+) T cells. Higher percentages of dividing CD4(+) and CD8(+) T cells were noted in patients with the higher viral burdens. No direct correlation was noted between rates of T cell turnover and CD4(+) T cell counts. Marked reductions in CD4(+) and CD8(+) T cell proliferation were seen in 11/11 patients 1-12 weeks after initiation of highly active antiretroviral therapy (HAART). These reductions persisted for the length of the study (16-72 weeks). Decreases in naive T cell proliferation correlated with increases in the levels of T cell receptor rearrangement excision circles. Division of CD4(+) and CD8(+) T cells increased dramatically in association with rapid increases in HIV-1 viral loads in 9/9 patients 5 weeks after termination of HAART and declined to pre-HAART-termination levels 8 weeks after reinitiation of therapy. These data are consistent with the hypothesis that HIV-1 infection induces a viral burden-related, global activation of the immune system, leading to increases in lymphocyte proliferation.

B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells.

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.

Effects of intermittent interleukin-2 therapy on plasma and tissue human immunodeficiency virus levels and quasi-species expression.

To characterize the effects of intermittent interleukin (IL)-2 therapy on human immunodeficiency virus (HIV), 11 patients underwent detailed virological evaluation during a year of IL-2 therapy. Six patients showed a >0.5 log increase in plasma HIV during at least 1 IL-2 cycle, with 2 experiencing an increase in >50% of cycles. Three of the remaining 5 patients had a >0.5 log decrease during at least 1 IL-2 cycle, and the remaining patients exhibited <0.5 log changes. No changes in lymphoid (tonsil) levels of HIV were seen during the year. Quasi-species analysis in a separate cohort demonstrated that the virus induced by IL-2 most commonly resembled pre-IL-2 plasma quasi species. Thus, intermittent IL-2 does not result in sustained increases in either plasma or tissue levels of HIV and does not result in sustained expression of a previously silent quasi species.

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression.

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4(+) T cell counts >/= 350 cells/microliter and viral load below the limits of detection for >/=1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2-3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log(10) copies) day(-1), which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log(10) copies) day(-1) (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4(+) T cells.

CD40-Mediated induction of CD4 and CXCR4 on B lymphocytes correlates with restricted susceptibility to human immunodeficiency virus type 1 infection: potential role of B lymphocytes as a viral reservoir.

Human immunodeficiency virus type 1 (HIV-1) replicates primarily in lymphoid tissues where it has ready access to activated immune competent cells. We used one of the major pathways of immune activation, namely, CD40-CD40L interactions, to study the infectability of B lymphocytes isolated from peripheral blood mononuclear cells. Highly enriched populations of B lymphocytes generated in the presence of interleukin-4 and oligomeric soluble CD40L upregulated costimulatory and activation markers, as well as HIV-1 receptors CD4 and CXCR4, but not CCR5. By using single-round competent luciferase viruses complemented with either amphotropic or HIV-derived envelopes, we found a direct correlation between upregulation of HIV-1 receptors and the susceptibility of the B lymphocytes to infection with dual-tropic and T-tropic strains of HIV-1; in contrast, cells were resistant to M-tropic strains of HIV-1. HIV-1 envelope-mediated infection was completely abolished with either an anti-CD4 monoclonal antibody or a peptide known to directly block CXCR4 usage and partially blocked with stromal cell-derived factor 1, all of which had no effect on the entry of virus pseudotyped with amphotropic envelope. Full virus replication kinetics confirmed that infection depends on CXCR4 usage. Furthermore, productive cycles of virus replication occurred rapidly yet under most conditions, without the appearance of syncytia. Thus, an activated immunological environment may induce the expression of HIV-1 receptors on B lymphocytes, priming them for infection with selective strains of HIV-1 and allowing them to serve as a potential viral reservoir.

Immunoreconstitution after ritonavir therapy in children with human immunodeficiency virus infection involves multiple lymphocyte lineages.

OBJECTIVE: To evaluate lymphocyte reconstitution after protease inhibitor therapy in children with human immunodeficiency virus (HIV) infection. STUDY DESIGN: Forty-four HIV-infected children receiving ritonavir monotherapy followed by the addition of zidovudine and didanosine were evaluated during a phase I/II clinical trial. The cohort had a median age of 6.8 years and advanced disease (57% Centers for Disease Control and Prevention stage C, 73% immune stage 3) and was naive to protease inhibitor therapy. RESULTS: After 4 weeks of therapy, there was a significant increase in CD4(+) and CD8(+) T cells. CD4(+) T cells continued to increase, whereas CD8(+) T cells returned to baseline by 24 weeks. Unexpectedly, there was a significant increase in B cells. Changes in CD4(+) T-cell subsets revealed an initial increase in CD4(+) CD45RO T cells followed by a sustained increase in CD4(+) CD45RA T cells. Children <6 years of age had the highest increase in all lymphocyte populations. Significant improvement in CD4(+) T-cell counts was observed even in those children whose viral burden returned to pre-therapy levels. CONCLUSIONS: Early increases in lymphocytes after ritonavir therapy are a result of recirculation, as shown by increases in B cells and CD4(+) CD45RO and CD8(+) T cells. Children exhibited a high potential to reconstitute CD4(+) CD45RA T cells even with advanced disease and incomplete viral suppression.

A randomized trial of high- versus low-dose subcutaneous interleukin-2 outpatient therapy for early human immunodeficiency virus type 1 infection.

Forty-nine outpatients infected with human immunodeficiency virus with baseline CD4 cell counts >/=500/mm3, who were on stable antiretroviral therapy, were randomized to receive 5-day cycles of either low-dose (1.5 million IU [MIU] twice a day) or high-dose (7.5 MIU twice a day) subcutaneous (sc) interleukin (IL)-2 every 4 or every 8 weeks. High-dose recipients experienced mean slopes of +116.1 cells/month and +2.7 %/month in CD4 cells and percents, respectively, whereas low-dose recipients displayed mean slopes of +26.7 and +1.3% in the same parameters. At month 6, high-dose recipients achieved a 94.8% increase in mean CD4 cells over baseline compared with a 19.0% increase in low-dose recipients. While high-dose recipients encountered more constitutional side effects, these were generally not dose-limiting. High-dose scIL-2 therapy in outpatients with early HIV-1 infection was well tolerated and induced dramatic, sustained rises in CD4 cells.

Pharmacokinetic modeling of recombinant interleukin-2 in patients with human immunodeficiency virus infection.

A novel model was developed to characterize the time-varying clearance of recombinant interleukin-2 (IL-2). Sixty-eight patients with human immunodeficiency virus infection received 83 cycles of IL-2 either by continuous infusion or by subcutaneous injection for 5 days. IL-2 concentrations after intravenous infusions peaked at 24 hours and then declined by 55% to 78% during the remainder of the infusion. Soluble IL-2 receptors increased greater than 10-fold before gradually returning to baseline. Subcutaneous administration showed a dose-dependent decrease in area under the concentration-time curve (AUC) between days 1 and 5. A model was developed in 9 patients who had IL-2 concentrations and soluble IL-2 receptors determined by ELISA. Concentrations were fitted by an indirect stimulatory pharmacodynamic model. An additional 59 patients with only IL-2 concentrations were fitted to a simplified empiric model. Both models provided an overall r2 of 0.99 for the plot of observed versus fitted concentrations. The time-dependent increase in IL-2 clearance, likely receptor-mediated, was well described with use of an indirect-effects pharmacokinetic-pharmacodynamic model.