Joseph M. McCune

Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia

BACKGROUND In many patients with human immunodeficiency virus (HIV) infection, therapy with potent antiretroviral drugs does not result in complete suppression of HIV replication. The effect of cessation of therapy in these patients is unknown. METHODS Sixteen patients who had a plasma HIV RNA level of more than 2500 copies per milliliter during combination antiretroviral-drug therapy were randomly assigned, in a 2:1 ratio, to discontinue or continue therapy. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility were measured weekly. Viral replicative capacity was measured at base line and at week 12. RESULTS Discontinuation of therapy for 12 weeks was associated with a median decrease in the CD4 cell count of 128 cells per cubic millimeter and an increase in the plasma HIV RNA level of 0.84 log copies per milliliter. Virus from all patients with detectable resistance at entry became susceptible to HIV-protease inhibitors within 16 weeks after the discontinuation of therapy. Drug susceptibility began to increase a median of six weeks after the discontinuation of therapy and was temporally associated with increases in plasma HIV RNA levels and decreases in CD4 cell counts. Viral replicative capacity, measured by means of a recombinant-virus assay, was low at entry into the study and increased after therapy was discontinued. Despite the loss of detectable resistance in plasma, resistant virus was cultured from peripheral-blood mononuclear cells in five of nine patients who could be evaluated. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility remained stable in the patients who continued therapy. CONCLUSIONS Despite the presence of reduced drug susceptibility, antiretroviral-drug therapy can provide immunologic and virologic benefit. This benefit reflects continued antiviral-drug activity and the maintenance of a viral population with a reduced replicative capacity.

Directly measured kinetics of circulating T lymphocytes in normal and HIV-1-infected humans

The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique1, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/μl per day and 6 CD8+ T cells/μl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/μl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.

The dynamics of CD4+ T-cell depletion in HIV disease.

The size and composition of the CD4+ T-cell population is regulated by balanced proliferation of progenitor cells and death of mature progeny. After infection with the human immunodeficiency virus, this homeostasis is often disturbed and CD4+ T cells are instead depleted. Such depletion cannot result simply from accelerated destruction of mature CD4+ T cells — sources of T-cell production must also fail. Ironically, this failure may be precipitated by physiological mechanisms designed to maintain homeostasis in the face of accelerated T-cell loss.

Relationship between T Cell Activation and CD4+ T Cell Count in HIV-Seropositive Individuals with Undetectable Plasma HIV RNA Levels in the Absence of Therapy

BACKGROUND Although untreated human immunodeficiency virus (HIV)-infected patients maintaining undetectable plasma HIV RNA levels (elite controllers) have high HIV-specific immune responses, it is unclear whether they experience abnormal levels of T cell activation, potentially contributing to immunodeficiency. METHODS We compared percentages of activated (CD38(+)HLA-DR(+)) T cells between 30 elite controllers, 47 HIV-uninfected individuals, 187 HIV-infected individuals with undetectable viremia receiving antiretroviral therapy (antiretroviral therapy suppressed), and 66 untreated HIV-infected individuals with detectable viremia. Because mucosal translocation of bacterial products may contribute to T cell activation in HIV infection, we also measured plasma lipopolysaccharide (LPS) levels. RESULTS Although the median CD4(+) cell count in controllers was 727 cells/mm(3), 3 (10%) had CD4(+) cell counts <350 cells/mm(3) and 2 (7%) had acquired immunodeficiency syndrome. Controllers had higher CD4(+) and CD8(+) cell activation levels (P < .001 for both) than HIV-negative subjects and higher CD8(+) cell activation levels than the antiretroviral therapy suppressed (P = .048). In controllers, higher CD4(+) and CD8(+) T cell activation was associated with lower CD4(+) cell counts (P = .009 and P = .047). Controllers had higher LPS levels than HIV-negative subjects (P < .001), and in controllers higher LPS level was associated with higher CD8(+) T cell activation (P = .039). CONCLUSION HIV controllers have abnormally high T cell activation levels, which may contribute to progressive CD4(+) T cell loss even without measurable viremia.

Increased production of IL-7 accompanies HIV-1–mediated T-cell depletion: implications for T-cell homeostasis

We hypothesized that HIV-1–mediated T-cell loss might induce the production of factors that are capable of stimulating lymphocyte development and expansion. Here we perform cross-sectional (n = 168) and longitudinal (n = 11) analyses showing that increased circulating levels of interleukin (IL)-7 are strongly associated with CD4+ T lymphopenia in HIV-1 disease. Using immunohistochemistry with quantitative image analysis, we demonstrate that IL-7 is produced by dendritic-like cells within peripheral lymphoid tissues and that IL-7 production by these cells is greatly increased in lymphocyte-depleted tissues. We propose that IL-7 production increases as part of a homeostatic response to T-cell depletion.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Tryptophan Catabolism by Indoleamine 2,3-Dioxygenase 1 Alters the Balance of TH17 to Regulatory T Cells in HIV Disease

Patients with AIDS have fewer immune cells to defend against microbial invasion through the gut, a critical loss that may be caused by a tryptophan metabolite produced by other immune cells. Loss of the Defenders at the Gate Like archers stationed along the walls of a medieval castle, the immune system patrols the vulnerable parts of our body to keep pathogens at bay. One of these susceptible areas is the mucosa of the gastrointestinal tract, which is continually exposed to ingested and resident pathogens. This defense breaks down in patients with AIDS, in which sentinel immune cells [T helper 17 (TH17) cells] are missing from the gastrointestinal lining, potentially accounting for some secondary infections acquired by these patients. Favre and colleagues present evidence that the loss of these cells (and a parallel increase in immune suppressor cells) is caused by a metabolite of the amino acid tryptophan, new understanding that should help to prevent this serious consequence of HIV infection. HIV disease is in part an inflammatory disease, and activated T cells and cytokines circulate in patients’ blood, along with pathogen-derived molecules that trigger the innate immune system. The authors show that, in patients with serious AIDS, who are in this inflammatory state, the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which catabolizes tryptophan, is elevated in dendritic cells (DCs)—agents that present antigen to the immune system—from the blood, lymph nodes, and mucosa of the lower gastrointestinal tract. The inflammation-related molecules interferon γ and bacterial lipopolysaccharide can induce IDO1 in isolated DCs. This excess IDO1 activation increased blood concentrations of tryptophan catabolites in patients, and two of the catabolites increased the proportion of TH17 (activating) immune cells and decreased the proportion of T regulatory (Treg) (suppressing) immune cells in culture. In patients with serious disease, the authors found that the ratio of TH17 to Treg cells was much lower than normal, which hampers the ability of the body to raise an effective immune defense against pathogens. This dysfunctional system would set up a reinforcing loop that progressively depletes vulnerable tissues of their immune protection. Paradoxically, it seems, activation of the immune system by HIV may be contributing to the decline in immune function that is the hallmark of the disease. IDO1 inhibitors are being tested for their efficacy in interfering with this dangerous depletion of defenses. The pathogenesis of human and simian immunodeficiency viruses is characterized by CD4+ T cell depletion and chronic T cell activation, leading ultimately to AIDS. CD4+ T helper (TH) cells provide protective immunity and immune regulation through different immune cell functional subsets, including TH1, TH2, T regulatory (Treg), and interleukin-17 (IL-17)–secreting TH17 cells. Because IL-17 can enhance host defenses against microbial agents, thus maintaining the integrity of the mucosal barrier, loss of TH17 cells may foster microbial translocation and sustained inflammation. Here, we study HIV-seropositive subjects and find that progressive disease is associated with the loss of TH17 cells and a reciprocal increase in the fraction of the immunosuppressive Treg cells both in peripheral blood and in rectosigmoid biopsies. The loss of TH17/Treg balance is associated with induction of indoleamine 2,3-dioxygenase 1 (IDO1) by myeloid antigen-presenting dendritic cells and with increased plasma concentration of microbial products. In vitro, the loss of TH17/Treg balance is mediated directly by the proximal tryptophan catabolite from IDO metabolism, 3-hydroxyanthranilic acid. We postulate that induction of IDO may represent a critical initiating event that results in inversion of the TH17/Treg balance and in the consequent maintenance of a chronic inflammatory state in progressive HIV disease.

Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism.

Gut-resident microbial populations may influence intestinal homeostasis during HIV disease. Gut Reaction to HIV Despite the value placed on individuality in the western world, no human is an island. Indeed, every person carries trillions of microorganisms in their intestine, and it’s becoming increasingly clear that the composition and interaction of these microorganisms can directly affect human health. Vujkovic-Cvijin et al. now demonstrate that this is the case for individuals infected with HIV, even those where viral load is controlled by therapy. The authors screened the intestinal microbiome for microbial drivers of HIV-associated immunopathology. They found that a dysbiotic mucosal microbial community associated with mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects, even in patients controlled with antiviral therapy. They then looked further into the mechanism of this association and found that the extent of dysbiosis correlated with tryptophan catabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Indeed, gut-resident bacteria with the capacity to metabolize tryptophan were found to be enriched in HIV-infected subjects. These data link gut-resident microbial populations with the immunopathogenesis of HIV and suggest that modulating this community could provide a new therapeutic strategy for managing HIV disease progression. Progressive HIV infection is characterized by dysregulation of the intestinal immune barrier, translocation of immunostimulatory microbial products, and chronic systemic inflammation that is thought to drive progression of disease to AIDS. Elements of this pathologic process persist despite viral suppression during highly active antiretroviral therapy (HAART), and drivers of these phenomena remain poorly understood. Disrupted intestinal immunity can precipitate dysbiosis that induces chronic inflammation in the mucosa and periphery of mice. However, putative microbial drivers of HIV-associated immunopathology versus recovery have not been identified in humans. Using high-resolution bacterial community profiling, we identified a dysbiotic mucosal-adherent community enriched in Proteobacteria and depleted of Bacteroidia members that was associated with markers of mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects. Furthermore, this dysbiosis was evident among HIV-infected subjects undergoing HAART, and the extent of dysbiosis correlated with activity of the kynurenine pathway of tryptophan catabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Gut-resident bacteria with capacity to catabolize tryptophan through the kynurenine pathway were found to be enriched in HIV-infected subjects, strongly correlated with kynurenine levels in HIV-infected subjects, and capable of kynurenine production in vitro. These observations demonstrate a link between mucosal-adherent colonic bacteria and immunopathogenesis during progressive HIV infection that is apparent even in the setting of viral suppression during HAART. This link suggests that gut-resident microbial populations may influence intestinal homeostasis during HIV disease.

Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of 2H2O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% 2H2O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0–200 μg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose–response relationship up to the 100 μg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose–response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the 2H2O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270–400 days and die-away values after 2H2O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1–1.5% new cells per day, whereas obese ad libitum-fed ob/ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term 2H2O enrichments in body water were achieved by daily 2H2O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body 2H2O enrichment (≈3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with 2H2O was 0.056 (CD4+) and 0.043 (CD8+) (replacement rate <0.1% per day). In summary, 2H2O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.

Factors influencing T-cell turnover in HIV-1–seropositive patients

HIV-1 disease is associated with pathological effects on T-cell production, destruction, and distribution. Using the deuterated (2H) glucose method for endogenous labeling, we have analyzed host factors that influence T-cell turnover in HIV-1-uninfected and -infected humans. In untreated HIV-1 disease, the average half life of circulating T cells was diminished without compensatory increases in cell production. Within 12 weeks of the initiation of highly active antiretroviral therapy (HAART), the absolute production rates of circulating T cells increased, and normal half-lives and production rates were restored by 12-36 months. Interpatient heterogeneity in the absolute degree of turnover correlated with the relative proportion of naive- and memory/effector-phenotype T cells in each of the CD4+ and CD8+ populations. The half-lives of naive-phenotype T cells ranged from 116-365 days (fractional replacement rates of 0.19-0.60% per day), whereas memory/effector-phenotype T cells persisted with half-lives from 22-79 days (fractional replacement rates of 0.87-3.14% per day). Naive-phenotype T cells were more abundant, and the half-life of total T cells was prolonged in individuals with abundant thymic tissue, as assessed by computed tomography. Such interpatient variation in T-cell kinetics may be reflective of differences in functional immune reconstitution after treatment for HIV-1 disease.