IL-21 and probiotic therapy improve Th17 frequencies, microbial translocation, and microbiome in ARV-treated, SIV-infected macaques.

Increased mortality in antiretroviral (ARV)-treated, HIV-infected individuals has been attributed to persistent immune dysfunction, in part due to abnormalities at the gastrointestinal barrier. In particular, the poor reconstitution of gastrointestinal Th17 cells correlates with residual translocation of dysbiotic, immunostimulatory microflora across a compromised intestinal epithelial barrier. We have previously demonstrated that oral probiotics promote increased intestinal CD4(+) T-cell reconstitution during ARV treatment in a non-human primate model of HIV infection; however, essential mucosal T-cell subsets, such as Th17 cells, had limited recovery. Here, we sought to promote Th17 cell recovery by administering interleukin (IL)-21 to a limited number of ARV-treated, probiotic-supplemented, Simian Immunodeficiency Virus (SIV)-infected pigtailed macaques. We demonstrate that probiotic and IL-21 supplementation of ARVs are associated with enhanced polyfunctional Th17 expansion and reduced markers of microbial translocation and dysbiosis as compared with infected controls receiving ARVs alone. Importantly, treatment resulted in fewer morbidities compared with controls, and was independent of increased immune activation or loss of viral suppression. We propose that combining ARVs with therapeutics aimed at restoring intestinal stasis may significantly improve disease prognosis of ARV-treated, HIV-infected individuals.

Variations in the heme oxygenase-1 microsatellite polymorphism are associated with plasma CD14 and viral load in HIV-infected African-Americans.

Heme oxygenase-1 (HO-1) is an anti-inflammatory enzyme that maintains homeostasis during cellular stress. Given previous findings that shorter length variants of a HO-1 promoter region GT(n) microsatellite polymorphism are associated with increased HO-1 expression in cell lines, we hypothesized that shorter variants would also be associated with increased levels of HO-1 expression, less inflammation and lower levels of inflammation-associated viral replication in human immunodeficiency virus (HIV)-infected subjects. Healthy donors (n = 20) with shorter GT(n) repeats had higher HO-1 mRNA transcript in peripheral blood mononuclear cells stimulated with lipopolysaccharide (r = -0.38, P = 0.05). The presence of fewer GT(n) repeats in subjects with untreated HIV disease was associated with higher HO-1 mRNA levels in peripheral blood (r = -0.41, P = 0.02); similar observations were made in CD14(+) monocytes from antiretroviral-treated subjects (r = -0.36, P = 0.04). In African-Americans, but not Caucasians, greater GT(n) repeats were correlated with higher soluble CD14 levels during highly active antiretroviral therapy (r = 0.38, P = 0.007) as well as higher mean viral load off-therapy (r = 0.24, P = 0.04). These data demonstrate that the HO-1 GT(n) microsatellite polymorphism is associated with higher levels of HO-1 expression and that this pathway may have important effects on the association between inflammation and HIV replication.

HLA class I-restricted T-cell responses may contribute to the control of human immunodeficiency virus infection, but such responses are not always necessary for long-term virus control.

A rare subset of human immunodeficiency virus (HIV)-infected individuals maintains undetectable HIV RNA levels without therapy ("elite controllers"). To clarify the role of T-cell responses in mediating virus control, we compared HLA class I polymorphisms and HIV-specific T-cell responses among a large cohort of elite controllers (HIV-RNA < 75 copies/ml), "viremic" controllers (low-level viremia without therapy), "noncontrollers" (high-level viremia), and "antiretroviral therapy suppressed" individuals (undetectable HIV-RNA levels on antiretroviral therapy). The proportion of CD4(+) and CD8(+) T cells that produce gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in response to Gag and Pol peptides was highest in the elite and viremic controllers (P < 0.0001). Forty percent of the elite controllers were HLA-B*57 compared to twenty-three percent of viremic controllers and nine percent of noncontrollers (P < 0.001). Other HLA class I alleles more common in elite controllers included HLA-B*13, HLA-B*58, and HLA-B*81 (P < 0.05 for each). Within elite and viremic controller groups, those with protective class I alleles had higher frequencies of Gag-specific CD8(+) T cells than those without these alleles (P = 0.01). Noncontrollers, with or without protective alleles, had low-level CD8(+) responses. Thus, certain HLA class I alleles are enriched in HIV controllers and are associated with strong Gag-specific CD8(+)IFN-gamma(+)IL-2(+) T cells. However, the absence of evidence of T cell-mediated control in many controllers suggests the presence of alternative mechanisms for viral control in these individuals. Defining mechanisms for virus control in "non-T-cell controllers" might lead to insights into preventing HIV transmission or preventing virus replication.

HIV-infected liver and kidney transplant recipients: 1- and 3-year outcomes.

Improvements in human immunodeficiency virus (HIV)-associated mortality make it difficult to deny transplantation based upon futility. Outcomes in the current management era are unknown. This is a prospective series of liver or kidney transplant recipients with stable HIV disease. Eleven liver and 18 kidney transplant recipients were followed for a median of 3.4 years (IQR [interquartile range] 2.9-4.9). One- and 3-year liver recipients' survival was 91% and 64%, respectively; kidney recipients' survival was 94%. One- and 3-year liver graft survival was 82% and 64%, respectively; kidney graft survival was 83%. Kidney patient and graft survival were similar to the general transplant population, while liver survival was similar to the older population, based on 1999-2004 transplants in the national database. CD4+ T-cell counts and HIV RNA levels were stable; and there were two opportunistic infections (OI). The 1- and 3-year cumulative incidence (95% confidence intervals [CI]) of rejection episodes for kidney recipients was 52% (28-75%) and 70% (48-92%), respectively. Two-thirds of hepatitis C virus (HCV)-infected patients, but no patient with hepatitis B virus (HBV) infection, recurred. Good transplant and HIV-related outcomes among kidney transplant recipients, and reasonable outcomes among liver recipients suggest that transplantation is an option for selected HIV-infected patients cared for at centers with adequate expertise.

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 (2)H(2)O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4% (2)H(2)O 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 microg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose-response relationship up to the 100 microg 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 (2)H(2)O 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 (2)H(2)O 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 obob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term (2)H(2)O enrichments in body water were achieved by daily (2)H(2)O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body (2)H(2)O enrichment ( approximately 3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with (2)H(2)O was 0.056 (CD4(+)) and 0.043 (CD8(+)) (replacement rate <0.1% per day). In summary, (2)H(2)O 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.

Poor CD4 T cell restoration after suppression of HIV-1 replication may reflect lower thymic function.

OBJECTIVE: To characterize immune phenotype and thymic function in HIV-1-infected adults with excellent virologic and poor immunologic responses to highly active antiretroviral therapy (HAART). METHODS: Cross-sectional study of patients with CD4 T cell rises of > or = 200 x 10(6) cells/l (CD4 responders; n = 10) or < 100 x 10(6) cells/l (poor responders; n = 12) in the first year of therapy. RESULTS: Poor responders were older than CD4 responders (46 versus 38 years; P < 0.01) and, before HAART, had higher CD4 cell counts (170 versus 35 x 106 cells/l; P = 0.11) and CD8 cell counts (780 versus 536 x 10(6) cells/l; P = 0.02). After a median of 160 weeks of therapy, CD4 responders had more circulating naive phenotype (CD45+CD62L+) CD4 cells (227 versus 44 x 10(6) cells/l; P = 0.001) and naive phenotype CD8 cells (487 versus 174 x 10(6) cells/l; P = 0.004) than did poor responders (after 130 weeks). Computed tomographic scans showed minimal thymic tissue in 11/12 poor responders and abundant tissue in 7/10 responders (P = 0.006). Poor responders had fewer CD4 cells containing T cell receptor excision circles (TREC) compared with CD4 responders (2.12 versus 27.5 x 10(6) cells/l; P = 0.004) and had shorter telomeres in CD4 cells (3.8 versus 5.3 kb; P = 0.05). Metabolic labeling studies with deuterated glucose indicated that the lower frequency of TREC-containing lymphocytes in poor responders was not caused by accelerated proliferation kinetics. CONCLUSION: Poor CD4 T cell increases observed in some patients with good virologic response to HAART may be caused by failure of thymic T cell production.

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.

Impaired replication of protease inhibitor-resistant HIV-1 in human thymus.

Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI 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.

Cytomegalovirus (CMV)-specific CD4+ T lymphocyte immune function in long-term survivors of AIDS-related CMV end-organ disease who are receiving potent antiretroviral therapy.

To better understand the relation of cytomegalovirus (CMV)-specific CD4+ T lymphocyte immunity and clinical outcome in AIDS-related CMV end-organ disease, 2 patient groups were prospectively studied: patients recently diagnosed with active CMV end-organ disease and survivors of CMV retinitis who had responded to highly active antiretroviral therapy and had quiescent retinitis when anti-CMV therapy was discontinued. Most patients with active CMV disease had negative CMV-specific CD4+ T lymphocyte responses at diagnosis, as measured by lymphoproliferation (7/7) or cytokine flow cytometry (3/5) assays. In contrast, all 10 subjects with quiescent retinitis and >150 absolute CD4+ T lymphocytes/microL whose anti-CMV therapy was discontinued during 6 months of follow-up had positive CMV-specific immune responses at least once by each assay. However, 6 of these 10 subjects also had negative CMV-specific immune responses > or =1 time. Such patients may be at risk for future CMV disease progression and should be closely monitored.

Loss of cytomegalovirus-specific CD4+ T cell responses in human immunodeficiency virus type 1-infected patients with high CD4+ T cell counts and recurrent retinitis.

Clinical histories are reported for 2 patients treated with highly active antiretroviral therapy (HAART) who experienced multiple relapses of cytomegalovirus (CMV) retinitis, despite suppression of human immunodeficiency virus type 1 (HIV-1) viremia and improvement in CD4+ T cell counts (to >400 cells/microL). CMV-specific CD4+ T cell immune reconstitution was measured directly, using cytokine flow cytometry, which revealed persistent deficits in CMV-specific CD4+ T cell responses in both patients. CMV-specific T cells constituted 0.14% and 0.05% of the total CD4+ T cell count in these patients, which is significantly lower than the percentages for 34 control subjects (0.6%-46%; CD4+ T cell count range, 7-1039 cells/microL; P=.019). Deficits in pathogen-specific immune responses may persist in some individuals, despite suppression of HIV-1 replication and substantial increases in circulating CD4+ T cells after HAART, and such deficits may be associated with significant morbidity from opportunistic infections.

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.

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects.

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.

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.

Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.

Causal relationships between HIV-1 coreceptor utilization, tropism, and pathogenesis in human thymus.

The pathogenic differences between CXCR4 (X4)- and CCR5 (R5)-utilizing strains of HIV-1 may be predominantly due to differences in viral tropism, which in turn may be due to differential coreceptor utilization. We tested this hypothesis in the human thymus organ of the SCID-hu Thy/Liv mouse, using recombinants of NL4-3 that were isogenic except for Env coreceptor-binding determinants of the V1-V3 loops. Conversion of NL4-3 from an X4 to an R5 isolate was associated with altered tropism for cell subpopulations within the Thy/Liv organ (with a higher frequency of infection of thymic stromal cells, including macrophages), a slower rate of replication, and a lower level of cytopathicity. These observations underscore the causal relationships between tropism, coreceptor use, and cytopathicity in the human thymus in vivo.

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.

A membrane-bound Fas decoy receptor expressed by human thymocytes.

Human thymocytes at several stages of maturation express Fas, yet resist apoptosis induction through its ligation. A proximal step in apoptotic signaling through Fas is implicated in this resistance, as these cells undergo normal levels of apoptosis induction after exposure to tumor necrosis factor-alpha. We studied the Fas receptors expressed in human thymocytes to search for mechanisms of receptor-mediated inhibition of Fas signaling in these cells. We describe here a unique, membrane-bound form of Fas receptor that contained a complete extracellular domain of Fas but that lacked a death domain due to alternative splicing of exon 7. This Fas decoy receptor (FDR) was shown to have nearly wild-type ability to bind native human Fas ligand and was expressed predominantly at the plasma membrane. Unlike soluble forms of Fas receptor, FDR dominantly inhibited apoptosis induction by Fas ligand in transfected human embryonic kidney cells. Titration of FDR in Fas-expressing cells suggests that FDR may operate through the formation of mixed receptor complexes. FDR also dominantly inhibited Fas-induced apoptosis in Jurkat T cells. In mixing experiments with wild-type Fas, FDR was capable of inhibiting death signaling at molar ratios less than 0.5, and this relative level of FDR:wild type message was observed in at least some thymocytes tested. The data suggest that Fas signal pathways in primary human cells may be regulated by expression of a membrane-bound decoy receptor, analogous to the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis by decoy receptors.