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2.  Myeloid-Lymphoid Ontogeny in the Rhesus Monkey (Macaca mulatta) 
Establishment of a functional immune system has important implications for health and disease, yet questions remain regarding the mechanism, location, and timing of development of myeloid and lymphoid cell compartments. The goal of this study was to characterize the ontogeny of the myeloid-lymphoid system in rhesus monkeys to enhance current knowledge of the developmental sequence of B cell (CD20, CD79), T cell (CD3, CD4, CD8, FoxP3), dendritic cell (CD205), and macrophage (CD68) lineages in the fetus and infant. Immunohistochemical assessments addressed the temporal and spatial expression of select phenotypic markers in the developing liver, thymus, spleen, lymph nodes, gut-associated lymphoid tissue (GALT), and bone marrow with antibodies known to cross-react with rhesus cells. CD3 was the earliest lymphoid marker identified in the first trimester thymus and, to a lesser extent, in the spleen. T cell markers were also expressed mid-gestation on cells of the liver, spleen, thymus, and in Peyer’s patches of the small and large intestine, and where CCR5 expression was noted. A myeloid marker, CD68, was found on hepatic cells near blood islands in the late first trimester. B cell markers were observed mid-second trimester in the liver, spleen, thymus, lymph nodes, bone marrow spaces, and occasionally in GALT. By the late third trimester and postnatally, secondary follicles with germinal centers were present in the thymus, spleen, and lymph nodes. These results suggest that immune ontogeny in monkeys is similar in temporal and anatomical sequence when compared to humans, providing important insights for translational studies.
doi:10.1002/ar.22943
PMCID: PMC4120262  PMID: 24867874
Fetus; Infant; Monkey; Immune System; Ontogeny
3.  The Kynurenine Pathway of Tryptophan Catabolism, CD4+ T-Cell Recovery, and Mortality Among HIV-Infected Ugandans Initiating Antiretroviral Therapy 
The Journal of Infectious Diseases  2014;210(3):383-391.
Background. Human immunodeficiency virus (HIV) infection–induced indoleamine 2,3-dioxygenase-1 (IDO) expression in activated monocytes and dendritic cells catabolizes tryptophan to kynurenine and other downstream catabolites that inhibit T-cell proliferation and interleukin 17 (IL-17) production. The prognostic significance of this pathway in treated HIV disease is unknown.
Methods. We measured systemic IDO activity (calculated as the ratio of plasma levels of kynurenine to tryptophan; hereafter, the “KT ratio”) in HIV-infected Ugandans before and during antiretroviral therapy (ART)–mediated viral suppression and its association with the rate of subsequent CD4+ T-cell count recovery and mortality.
Results. Among 435 participants, a higher pre-ART KT ratio was associated with a higher plasma virus load (P < .001) and lipopolysaccharide level (P = .018), a lower CD4+ T-cell count (P < .001), and female sex (P = .047). Through month 12 of ART-mediated viral suppression, the plasma KT ratio decreased by approximately 50% (P < .001). After adjustment for pre-ART CD4+ T-cell count, virus load, age, and sex, a higher month 12 KT ratio predicted a slower rate of subsequent CD4+ T-cell count recovery (P = .001). Thirty-nine participants died. After adjustment for pre-ART CD4+ T-cell count, virus load, body mass index, sex, and age, a higher pre-ART and month 6 KT ratio predicted increased mortality (P ≤ .016).
Conclusions. The kynurenine pathway of tryptophan catabolism independently predicts poor CD4+ T-cell count recovery and increased mortality among HIV-infected Ugandans initiating ART and may be an important target for interventions.
doi:10.1093/infdis/jiu115
PMCID: PMC4148610  PMID: 24585899
Tryptophan; kynurenine; indoleamine 2,3-dioxygenase-1; HIV; mortality; antiretroviral therapy; Uganda
4.  Low Proportions of CD28− CD8+ T cells Expressing CD57 Can Be Reversed by Early ART Initiation and Predict Mortality in Treated HIV Infection 
The Journal of Infectious Diseases  2014;210(3):374-382.
Background. Unlike cytomegalovirus (CMV) infection and aging, human immunodeficiency virus (HIV) decreases the proportion of CD28−CD8+ T cells expressing CD57. Whether this abnormality predicts mortality in treated HIV infection and can be reversed by early antiretroviral therapy (ART) remains unknown.
Methods. We sampled recently HIV-infected individuals (<6 months) and HIV-uninfected controls and compared longitudinal changes in the proportion of CD28−CD8+ T cells expressing CD57 between those who initiated ART early (<6 months) vs later (≥2 years). We also assessed the relationship between this phenotype and mortality in a nested case-control study of ART-suppressed chronically infected individuals.
Results. Compared to HIV-uninfected controls (n = 15), individuals who were recently infected with HIV had lower proportions of CD28−CD8+ T cells expressing CD57 (P < .001), and these proportions increased during ART. The early ART group (n = 33) achieved normal levels, whereas the later ART group (n = 30) continued to have lower levels than HIV-uninfected controls (P = .02). Among 141 ART-suppressed participants in the SOCA study, those in the lowest quartile of CD28−CD8+ T cells expressing CD57 had 5-fold higher odds of mortality than those in the highest quartile (95% CI, 1.6–15.9, P = .007).
Conclusions. Abnormally low proportions of CD28−CD8+ T cells expressing CD57 predict increased mortality during treated HIV infection and may be reversed with early ART initiation.
doi:10.1093/infdis/jiu109
PMCID: PMC4110459  PMID: 24585893
HIV; CD57; CD28; Immunosenescence; aging; mortality; antiretroviral therapy; immune activation
5.  Preclinical Evaluation of HIV Eradication Strategies in the Simian Immunodeficiency Virus-Infected Rhesus Macaque: A Pilot Study Testing Inhibition of Indoleamine 2,3-Dioxygenase 
Abstract
Even in the setting of maximally suppressive antiretroviral therapy (ART), HIV persists indefinitely. Several mechanisms might contribute to this persistence, including chronic inflammation and immune dysfunction. In this study, we have explored a preclinical model for the evaluation of potential interventions that might serve to eradicate or to minimize the level of persistent virus. Given data that metabolic products of the inducible enzyme indoleamine 2,3-dioxygeanse (IDO) might foster inflammation and viral persistence, chronically simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques were treated with the IDO inhibitor 1-methyl tryptophan (1mT). Orally administered 1mT achieved targeted plasma levels, but did not impact tryptophan metabolism or decrease viral RNA or DNA in plasma or in intestinal tissues beyond levels achieved by ART alone. Animals treated with 1mT showed no difference in the levels of T cell activation or differentiation, or in the kinetics or magnitude of viral rebound following cessation of ART. Notwithstanding these negative results, our observations suggest that the chronically SIV-infected rhesus macaque on suppressive ART can serve as a tractable model in which to test and to prioritize the selection of other potential interventions designed to eradicate HIV in vivo. In addition, this model might be used to optimize the route and dose by which such interventions are administered and the methods by which their effects are monitored.
doi:10.1089/aid.2012.0162
PMCID: PMC3552181  PMID: 22924680
6.  Induction of MHC Class I Expression on Immature Thymocytes in HIV-1-Infected SCID-hu Thy/Liv Mice: Evidence of Indirect Mechanisms1 
The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.
PMCID: PMC4435947  PMID: 10358212
7.  Monocyte Activation by Interferon α Is Associated With Failure to Achieve a Sustained Virologic Response After Treatment for Hepatitis C Virus Infection 
The Journal of Infectious Diseases  2013;209(10):1602-1612.
Background. Interferon α (IFN-α) and ribavirin can induce a sustained virologic response (SVR) in some but not all hepatitis C virus (HCV)–infected patients. The mechanism of effective treatment is unclear. One possibility is that IFN-α differentially improves the functional capacity of classic myeloid dendritic cells (mDCs) by altering expression of surface molecules or cytokines. Others have proposed that antigen-presenting cell activation could be paradoxically detrimental during HCV infection because of the production by monocytes of substances inhibitory or toxic to plasmacytoid dendritic cells.
Methods. We examined responses to in vitro IFN-α treatment of peripheral blood leukocyte samples from a retrospective treatment cohort of nearly 200 HCV-seropositive patients who had undergone antiviral therapy with ribavirin and pegylated IFN. We analyzed the variable responses of antigen-presenting cell subsets to drug.
Results. We found that patients achieving SVR were no more likely to have robust mDC activation in response to IFN-α than those who did not achieve SVR. Rather, patients achieving SVR were distinguished by restrained monocyte activation in the presence of IFN-α, a factor that was second in importance only to IL28B genotype in its association with SVR.
Conclusions. These results suggest that interindividual variability in the response of monocytes to IFN-α is an important determinant of treatment success with IFN-α–based regimens.
doi:10.1093/infdis/jit801
PMCID: PMC3997582  PMID: 24325966
hepatitis C virus; interferon-α; monocytes; activation; dendritic cells; sustained virologic response; treatment; ribavirin; Toll-like receptor 2
8.  A functional variant in FCRL3 is associated with higher FcRL3 expression on T cell subsets and rheumatoid arthritis disease activity 
Arthritis and rheumatism  2012;64(8):2451-2459.
Objective
CD4+FoxP3+ regulatory T cells (Treg) suppress effector T cells and prevent autoimmune disease. Treg function is deficient in active rheumatoid arthritis (RA), a loss which may play a role in the pathogenesis of this disease. We previously showed that a single nucleotide polymorphism in the Fc Receptor Like-3 (FCRL3) gene led to higher expression of FcRL3 on Treg+ and that FcRL3 Treg are functionally deficient in comparison to FcRL3− Treg. The objective of this work was to investigate the potential role of FcRL3 in rheumatoid arthritis.
Methods
A cross-sectional study was performed to evaluate the FCRL3 -169 genotype and FcRL3 expression on T cell subsets, including Treg, from peripheral blood samples of 51 patients with RA enrolled in the UCSF RA Cohort. Clinical data were obtained from the UCSF RA Cohort database.
Results
We found that patients with the FCRL3 -169C allele (genotype C/C or C/T) expressed significantly higher levels of FcRL3 on Treg, and on CD8+ and TCRγδ+ T cells, in comparison to RA patients with the T/T genotype. Higher FcRL3 expression on these T cell subpopulations correlated with RA disease activity in those patients harboring the FCRL3 -169C allele. Furthermore, FcRL3 expression on Treg was higher in patients with erosive RA disease and the FCRL3 -169C allele was overrepresented in patients with erosive RA disease.
Conclusion
FcRL3 expression, which is strongly associated with the presence of the FCRL3 -169C allele, may serve as a biomarker of RA disease activity.
doi:10.1002/art.34457
PMCID: PMC3374037  PMID: 22392608
9.  The Immunologic Effects of Mesalamine in Treated HIV-Infected Individuals with Incomplete CD4+ T Cell Recovery: A Randomized Crossover Trial 
PLoS ONE  2014;9(12):e116306.
The anti-inflammatory agent, mesalamine (5-aminosalicylic acid) has been shown to decrease mucosal inflammation in ulcerative colitis. The effect of mesalamine in HIV-infected individuals, who exhibit abnormal mucosal immune activation and microbial translocation (MT), has not been established in a placebo-controlled trial. We randomized 33 HIV-infected subjects with CD4 counts <350 cells/mm3 and plasma HIV RNA levels <40 copies/ml on antiretroviral therapy (ART) to add mesalamine vs. placebo to their existing regimen for 12 weeks followed by a 12 week crossover to the other arm. Compared to placebo-treated subjects, mesalamine-treated subjects did not experience any significant change in the percent CD38+HLA-DR+ peripheral blood CD4+ and CD8+ T cells at week 12 (P  = 0.38 and P  = 0.63, respectively), or in the CD4+ T cell count at week 12 (P  = 0.83). The percent CD38+HLA-DR+ CD4+ and CD8+ T cells also did not change significantly in rectal tissue (P  = 0.86, P  = 0.84, respectively). During the period of mesalamine administration, plasma sCD14, IL-6, D-dimer, and kynurenine to tryptophan ratio were not changed significantly at week 12 and were similarly unchanged at week 24. This study suggests that, at least under the conditions studied, the persistent immune activation associated with HIV infection is not impacted by the anti-inflammatory effects of mesalamine.
Trial Registration
ClinicalTrials.gov NCT01090102
doi:10.1371/journal.pone.0116306
PMCID: PMC4283685  PMID: 25545673
10.  Naïve human T cells are activated and proliferate in response to the heme oxygenase-1 (HO-1) inhibitor tin mesoporphyrin 
Heme oxygenase-1 (HO-1) and its catabolic byproducts have potent anti-inflammatory activity in many models of disease. It is not known, however, if HO-1 also plays a role in the homeostatic control of T cell activation and proliferation. We demonstrate here that the HO-1 inhibitor, tin mesoporphyrin (SnMP), induces activation, proliferation, and maturation of naïve CD4+ and CD8+ T cells via interactions with CD14+ monocytes in vitro. This response is dependent upon interactions of T cells with MHC Class I and II on the surface of CD14+ monocytes. Furthermore, CD4+CD25+FoxP3+ regulatory T cells (Tregs) were able to suppress this proliferation, even though their suppressive activity was itself impaired by SnMP. Given the magnitude of the antigen-independent T cell response induced by SnMP, we speculate that HO-1 plays an important role in dampening non-specific T cell activation. Based on these findings, we propose a potential role for HO-1 in the control of naïve T cell homeostatic proliferation.
doi:10.4049/jimmunol.0903127
PMCID: PMC3314532  PMID: 20921523
11.  Increase in 2–Long Terminal Repeat Circles and Decrease in D-dimer After Raltegravir Intensification in Patients With Treated HIV Infection: A Randomized, Placebo-Controlled Trial 
The Journal of Infectious Diseases  2013;208(9):1436-1442.
Background. The degree to which human immunodeficiency virus (HIV) continues to replicate during antiretroviral therapy (ART) is controversial. We conducted a randomized, double-blind, placebo-controlled study to assess whether raltegravir intensification reduces low-level viral replication, as defined by an increase in the level of 2–long terminal repeat (2-LTR) circles.
Methods. Thirty-one subjects with an ART-suppressed plasma HIV RNA level of <40 copies/mL and a CD4+ T-cell count of ≥350 cells/mm3 for ≥1 year were randomly assigned to receive raltegravir 400 mg twice daily or placebo for 24 weeks. 2-LTR circles were analyzed by droplet digital polymerase chain reaction at weeks 0, 1, 2, and 8.
Results. The median duration of ART suppression was 3.8 years. The raltegravir group had a significant increase in the level of 2-LTR circles, compared to the placebo group. The week 1 to 0 ratio was 8.8-fold higher (P = .0025) and the week 2 to 0 ratio was 5.7-fold higher (P = .023) in the raltegravir vs. placebo group. Intensification also led to a statistically significant decrease in the D-dimer level, compared to placebo (P = .045).
Conclusions. Raltegravir intensification resulted in a rapid increase in the level of 2-LTR circles in a proportion of subjects, indicating that low-level viral replication persists in some individuals even after long-term ART. Intensification also reduced the D-dimer level, a coagulation biomarker that is predictive of morbidity and mortality among patients receiving treatment for HIV infection.
doi:10.1093/infdis/jit453
PMCID: PMC3789577  PMID: 23975885
HIV; raltegravir intensification; 2-LTR circles; ongoing viral replication; D-dimer
12.  Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans 
Science (New York, N.y.)  2010;330(6011):1695-1699.
Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or “layering”) of unique hematopoietic stem cells (HSC) that give rise to distinct lymphocyte lineages at different stages of development. Here, we provide evidence of an analogous “layered” immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSC present at different stages of development. We also provide evidence that the fetal T cell lineage is biased towards immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.
doi:10.1126/science.1196509
PMCID: PMC3276679  PMID: 21164017
13.  Programmed death-1 expression on CD4+ and CD8+ T cells in treated and untreated HIV disease 
AIDS (London, England)  2014;28(12):1749-1758.
Background
There is intense interest in the role of programmed death 1 (PD-1) in causing persistent T-cell dysfunction in HIV infection. However, the impact of HIV infection and antiretroviral treatment (ART) on the expression of PD-1 on T cells is still poorly defined.
Methods
PD-1 was measured longitudinally in a cohort of recently HIV-infected individuals (n = 121) who started ART early (<6 months after infection) vs. later (≥2 years after infection). PD-1 was also measured cross-sectionally in a diverse cohort of chronically HIV-infected adults (n = 206).
Results
PD-1 expression levels were high on CD8+ T cells during early HIV infection. PD-1 levels increased on both CD4+ and CD8+ T cells populations in those who delayed therapy (11 and 10%/year, respectively). PD-1 levels declined and were similar in those treated early vs. late after 1 year of ART. In both cohorts, PD-1 expression on CD4+ T cells was associated with CD4+ T-cell activation (CD38+HLA-DR+) and inversely with CD4+ cell count. In contrast, PD-1 expression on CD8+ T cells was most strongly associated with CD8+ T-cell activation and with plasma viral load in viremic individuals.
Conclusion
Across two large cohorts of untreated and treated individuals, we found consistent associations between HIV RNA levels, CD8+ T-cell activation and PD-1 expression on CD8+ T cells. In contrast, CD4+ T-cell counts and CD4+ T-cell activation were more consistent correlates of PD-1 expression on CD4+ T cells. PD-1 expression appears to be driven by both direct antigen and homeostatic pathways.
doi:10.1097/QAD.0000000000000314
PMCID: PMC4206412  PMID: 24871455
CD4+ lymphocyte count; early antiretroviral therapy; HIV antiretroviral therapy; HIV-1/immunology/*physiology; humans; programmed death-1; T-cell activation; T lymphocytes/immunology/*physiology; virus replication/physiology
14.  The Human Fetal Immune Response to Hepatitis C Virus Exposure in Utero 
The Journal of Infectious Diseases  2011;203(2):196-206.
Background. Although the rate of mother-to-child transmission of hepatitis C virus (HCV) is low, the effect of HCV exposure in utero on the fetal immune system is unknown.
Methods. Umbilical cord blood was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women and 8 neonates born to HCV-seronegative women. Cord blood mononuclear cells were analyzed by immunophenotyping and by intracellular cytokine staining after HCV-specific and polyclonal stimulation. Plasma was analyzed for anti-HCV immunoglobulin M (IgM), cytokine/granzyme concentrations, and indoleamine 2,3-dioxygenase (IDO) activity.
Results. HCV-exposed neonates had significantly lower levels of regulatory T cells expressing HLA-DR, lower CD4+ and CD8+ T cell activation, and lower plasma levels of pro-inflammatory markers than did controls. However, CD4+ and CD8+ T cells from HCV-exposed neonates had higher IFN-γ production in response to polyclonal stimulation than did T cells from controls. IDO activity was similar between groups. No HCV-specific T cell responses or anti-HCV IgM were detected in any neonates.
Conclusions. HCV-exposed neonates showed a relative suppression of immune activation and pro-inflammatory markers, which was counterbalanced by an increased production capacity for IFN-γ. These results suggest that HCV encounters the fetal immune system in utero, and alters the balance between suppressive and pro-inflammatory responses.
doi:10.1093/infdis/jiq044
PMCID: PMC3071071  PMID: 21288819
15.  Antiretroviral Therapy Initiated Within 6 Months of HIV Infection Is Associated With Lower T-Cell Activation and Smaller HIV Reservoir Size 
The Journal of Infectious Diseases  2013;208(8):1202-1211.
Background. CD4+/CD8+ T-cell activation levels often remain elevated in chronic human immunodeficiency virus (HIV) infection despite initiation of antiretroviral therapy (ART). T-cell activation predicts early death and blunted CD4+ T-cell recovery during ART and may affect persistent HIV reservoir size. We investigated whether very early ART initiation is associated with lower on-therapy immune activation and HIV persistence.
Methods. From a cohort of patients with early HIV infection (<6 months duration since infection) we identified persons who started ART early (<6 months after infection) or later (≥2 years after infection) and maintained ≥2 years of virologic suppression; at-risk HIV-negative persons were controls. We measured CD4+/CD8+ T-cell activation (percent CD38+/HLA-DR+) and HIV reservoir size (based on HIV DNA and cell-associated RNA levels).
Results. In unadjusted analyses, early ART predicted lower on-therapy CD8+ T-cell activation (n = 34; mean, 22.1%) than achieved with later ART (n = 32; mean, 28.8%; P = .009), although levels in early ART remained elevated relative to HIV-negative controls (P = .02). Early ART also predicted lower CD4+ T-cell activation than with later ART (5.3% vs 7.5%; P = .06). Early ART predicted 4.8-fold lower DNA levels than achieved with later ART (P = .005), and lower cell-associated RNA levels (difference in signal-to-cutoff ratio (S/Co), 3.2; P = .035).
Conclusions. ART initiation <6 months after infection is associated with lower levels of T-cell activation and smaller HIV DNA and RNA reservoir size during long-term therapy.
doi:10.1093/infdis/jit311
PMCID: PMC3778965  PMID: 23852127
HIV antiretroviral therapy; early ART; T-cell activation; inflammation; HIVreservoir; HIV eradication; HIV cure
16.  Expression of the autoimmune susceptibility gene FcRL3 on human regulatory T cells is associated with dysfunction and high levels of PD-1 
CD4+FoxP3+ regulatory T cells (Treg) play a critical role in maintaining self tolerance and inhibiting autoimmune disease. Despite being a major focus of modern immunological investigation, many aspects of Treg biology remain unknown. In a screen for novel candidate genes involved in human Treg function, we detected the expression of an autoimmune susceptibility gene, FcRL3, in Treg but not in conventional CD4+ T cells. FcRL3 is an orphan receptor of unknown function with structural homology to classical Fc receptors. Numerous genetic studies have demonstrated a link between a single nucleotide polymorphism in the FCRL3 promoter and both overexpression of FcRL3 and autoimmune diseases such as rheumatoid arthritis. Given the critical role of Treg in suppressing autoimmunity, we sought to ascertain how expression of FcRL3 relates to the phenotype, differentiation, and function of Treg. We show here that FcRL3 is expressed on a population of thymically derived Treg that exhibits a memory phenotype and high levels of programmed cell death-1 (PD-1). Purified FcRL3+ Treg are less responsive to antigenic stimulation in the presence of IL-2 than their FcRL3− counterparts, despite intact proximal and distal IL-2 signaling as determined by phosphorylation of Stat-5 and upregulation of Bcl2. In vitro suppression assays demonstrated that FcRL3+ Treg have reduced capacity to suppress the proliferation of effector T cells. These data suggest that FcRL3 expression is associated with Treg dysfunction that may, in turn, contribute to the loss of self tolerance and the development of autoimmunity.
doi:10.4049/jimmunol.0903943
PMCID: PMC2894810  PMID: 20190142
17.  Levels of circulating myeloid subpopulations and of heme oxygenase-1 do not predict CD4+ T cell recovery after the initiation of antiretroviral therapy for HIV disease 
The level (or frequency) of circulating monocyte subpopulations such as classical (CD14hiCD16-) and non-classical (CD14dimCD16+) monocytes varies during the course of HIV disease progression and antiretroviral therapy (ART). We hypothesized that such variation and/or differences in the degree to which these cells expressed the immunoregulatory enzyme, heme oxygenase-1 (HO-1), would be associated with CD4+ T cell recovery after the initiation of ART. This hypothesis was tested in a cross-sectional study of four groups of HIV-infected subjects, including those who were seronegative, untreated virologic controllers [detectable viral load (VL) of <1000 copies/mL], untreated virologic non-controllers [VL > 10,000 copies/mL], and ART-mediated virologic controllers [VL < 75 copies/mL]. A longitudinal analysis of ART-treated subjects was also performed along with regression analysis to determine which biomarkers were associated with and/or predictive of CD4+ T cell recovery. Suppressive ART was associated with increased levels of classical monocyte subpopulations (CD14hiCD16-) and decreased levels of non-classical monocyte populations (CD14dimCD16+). Among peripheral blood mononuclear cells (PBMCs), HO-1 was found to be most highly up-regulated in CD14+ monocytes after ex vivo stimulation. Neither the levels of monocyte subpopulations nor of HO-1 expression in CD14+ monocytes were significantly associated with the degree of CD4+ T cell recovery. Monocyte subpopulations and HO-1 gene expression were, however, restored to normal levels by suppressive ART. These results suggest that the level of circulating monocyte subpopulations and their expression of HO-1 have no evident relationship to CD4+ T cell recovery after the initiation of ART.
doi:10.1186/1742-6405-11-27
PMCID: PMC4150425  PMID: 25180041
HIV; HO-1; Immune activation; Monocytes; CD4+ T cell recovery
18.  IL-7 production in murine lymphatic endothelial cells and induction in the setting of peripheral lymphopenia 
International Immunology  2013;25(8):471-483.
IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.
doi:10.1093/intimm/dxt012
PMCID: PMC3720065  PMID: 23657000
endothelium; HIV; interleukin-7; lymphatic; lymph node; lymphopenia; myeloid; stromal
19.  Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism 
Science translational medicine  2013;5(193):193ra91.
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 metabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Gut-resident bacteria with capacity to metabolize 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, which 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.
doi:10.1126/scitranslmed.3006438
PMCID: PMC4094294  PMID: 23843452
20.  Th17 and regulatory T cells: implications for AIDS pathogenesis 
Current opinion in HIV and AIDS  2010;5(2):151-157.
Purpose of review
The present review discusses recent reports showing that reciprocal changes in T helper interleukin-17-secreting CD4+ Th17 cells and CD4+CD25highFoxP3+ regulatory T cells (Tregs) may play a role in the progressive disease caused by the HIV and by simian immunodeficiency virus.
Recent findings
Studies in nonhuman primate models of lentiviral infection and in HIV-infected human individuals have shown that pathogenic infection is associated with loss of Th17 cells and an increase in the frequency of Tregs. Because interleukin-17 serves to maintain the integrity of the mucosal barrier, loss of Th17 cells may permit the increase in microbial translocation across the gastrointestinal mucosa that is observed in pathogenic lentiviral disease. It remains unclear, however, whether Th17 cells are preferentially infected or if, instead, their loss is induced by bystander effects of lentiviral infection, for example, the induction of indoleamine 2,3-dioxygenase.
Summary
Progressive lentiviral disease is associated with preferential depletion of Th17 cells and loss of Th17/Treg balance. Further analysis of such changes in the composition of subset CD4+ T helper and Tregs may shed new light on the immunopathology of HIV disease and suggest new strategies for therapeutic and preventive interventions.
doi:10.1097/COH.0b013e328335c0c1
PMCID: PMC2999911  PMID: 20543593
HIV; indoleamine 2,3-dioxygenase; regulatory T cells; Th17 cells
21.  Th17 cells and regulatory T cells in elite control over HIV and SIV 
Current opinion in HIV and AIDS  2011;6(3):221-227.
Purpose of review
We present current findings about two subsets of CD4+ T cells that play an important part in the initial host response to infection with the human immunodeficiency virus type 1 (HIV): those producing IL-17 (Th17 cells) and those with immunosuppressive function (CD25+FoxP3+ regulatory T cells, or T-reg). The role of these cells in control of viral infection and immune activation as well as in prevention of immune deficiency in HIV-infected elite controllers will be examined. We will also discuss the use of the simian immunodeficiency virus (SIV)-infected macaque model of AIDS to study the interplay between these cells and lentiviral infection in vivo.
Recent Findings
Study of Th17 cells in humans and non-human primates (NHP) has shown that depletion of these cells is associated with dissemination of microbial products from the infected gut, increased systemic immune activation, and disease progression. Most impressively, having a smaller Th17 cell compartment has been found to predict these outcomes. T-reg have been associated with reduced antiviral T cell responses but not with suppression of generalized T cell activation. Both cell subsets influence innate immune responses and, in so doing, may shape the inflammatory milieu of the host at infection.
Summary
Interactions between Th17 cells, T-reg, and cells of the innate immune system influence the course of HIV and SIV infection from its earliest stages, even before the appearance of adaptive immunity. Such interactions may be pivotal for elite control over disease progression.
doi:10.1097/COH.0b013e32834577b3
PMCID: PMC4079838  PMID: 21399494
HIV; SIV; Th17; regulatory T cells; T-reg; elite
22.  Cell-Based Measures of Viral Persistence Are Associated With Immune Activation and Programmed Cell Death Protein 1 (PD-1)–Expressing CD4+ T cells 
Background. Studies aimed at defining the association between host immune responses and human immunodeficiency virus (HIV) persistence during therapy are necessary to develop new strategies for cure.
Methods. We performed a comprehensive assessment of ultrasensitive plasma HIV RNA levels, cell-associated HIV RNA levels, proviral HIV DNA levels, and T cell immunophenotyping in a cohort of 190 subjects in whom HIV levels were suppressed by highly active antiretroviral therapy.
Results. The median CD4+ T cell count was 523 cells/mm3, and the median duration of viral suppression was 31 months. Cell-associated RNA and proviral DNA levels (but not ultrasensitive plasma HIV RNA levels) were positively correlated with frequencies of CD4+ and CD8+ T cells expressing markers of T-cell activation/dysfunction (CD38, HLA-DR, CCR5, and/or programmed cell death protein 1 [PD-1]) (P < .05). Having a low CD4+ T-cell count despite receipt of virologically suppressive therapy was associated with high cell-associated RNA and proviral DNA levels (P < .01) and higher frequencies of CD4+ T cells expressing CD38, HLA-DR, CCR5, and/or PD-1 (P < .0001).
Conclusions. Cell-based measurements of viral persistence were consistently associated with markers of immune activation and the frequency of PD-1–expressing CD4+ T cells. Treated patients with a low CD4+ T-cell count had higher frequencies of PD-1–expressing CD4+ T cells and cell-based measures of viral persistence, suggesting that HIV infection in these individuals may be more difficult to cure and may require unique interventions.
doi:10.1093/infdis/jis630
PMCID: PMC3666131  PMID: 23089590
HIV; raltegravir intensification; 2-LTR circles; ongoing viral replication; D-dimer
23.  HIV-Infected Individuals with Low CD4/CD8 Ratio despite Effective Antiretroviral Therapy Exhibit Altered T Cell Subsets, Heightened CD8+ T Cell Activation, and Increased Risk of Non-AIDS Morbidity and Mortality 
PLoS Pathogens  2014;10(5):e1004078.
A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm3) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.
Author Summary
The CD4/CD8 ratio, a hallmark of the collection of T cell defects related to aging –“immunosenescence”- and a predictor of mortality in the general population, often fails to normalize in an important proportion of HIV-infected individuals with adequate CD4+ T cell recovery after ART initiation. However, the immunological and clinical characteristics of this clinical phenotype have not been elucidated. Herein we show that during treated HIV infection, expansion of CD8+ T cells, reflected as a low CD4/CD8 ratio, identifies a subgroup of individuals with a number of immunological abnormalities and a poor prognosis. These subjects exhibit increased innate and adaptive immune activation, an immunosenescent phenotype, CD4+ and CD8+ imbalance in the gut mucosa and higher risk of morbidity and mortality. In contrast, those who normalize the CD4/CD8 ratio have traits of a healthy immune system. We observed that early ART initiation might contribute to more rapid and robust CD4/CD8 ratio normalization compared to later initiation. Hence, the CD4/CD8 ratio might help to further discriminate the risk of disease progression of successfully treated HIV-infected individuals, and a successful response to ART may require both normalization of the peripheral CD4+ T cell count and the ratio of CD4+ to CD8+ T cell counts.
doi:10.1371/journal.ppat.1004078
PMCID: PMC4022662  PMID: 24831517
24.  Central memory CD8+ T cells have a shorter lifespan and reduced abundance as a function of HIV disease progression1 
Progressive HIV disease has been associated with loss of memory T cell responses to antigen. To better characterize and quantify long-lived memory T cells in vivo, we have refined an in vivo labeling technique to study the kinetics of phenotypically distinct, low frequency CD8+ T cell subpopulations in humans. HIV-negative subjects and antiretroviral-untreated HIV-infected subjects in varying stages of HIV disease were studied. After labeling the DNA of dividing cells with deuterated water (2H2O), 2H-label incorporation and die-away kinetics were quantified using a highly sensitive FACS/mass spectrometric method. Two different populations of long-lived memory CD8+ T cells were identified in HIV-negative subjects: CD8+CD45RA-CCR7+CD28+ central memory (TCM) cells expressing IL-7Rα and CD8+CD45RA+CCR7-CD28- RA effector memory (TEmra) cells expressing CD57. In pilot studies in HIV-infected subjects, TCm cells were found to have a shorter half-life and reduced abundance, particularly in those with high viral loads; TEmra cells, by contrast, retained a long half-life and accumulated in the face of progressive HIV disease. These data are consistent with the hypothesis that IL-7Rα+ TCm cells represent “true” memory CD8+ T cells, the loss of which may be responsible in part for the progressive loss of T cell memory function during progressive HIV infection.
PMCID: PMC2562545  PMID: 18523254
Human; T cells; AIDS; Cell proliferation; Memory
25.  HIV-Induced Changes in T Cell Signaling Pathways1 
Infection with HIV usually results in chronic activation of the immune system, with profound quantitative and qualitative changes in the T cell compartment. To better understand the mechanistic basis for T cell dysfunction and to discern whether such mechanisms are reversed after effective antiviral treatment, we analyzed changes in signaling pathways of human CD4+ and CD8+ T cells from 57 HIV-infected subjects in varying stages of disease progression and treatment, including long-term nonprogressors, progressors, and chronically infected subjects provided effective antiretroviral therapy (responders). A previously described PhosFlow method was adapted and optimized so that protein phosphorylation could be visualized in phenotypically defined subpopulations of CD4+ and CD8+ T cells (naive, memory, and effector) by flow cytometry. T cell signaling induced by TCR cross-linking, IL-2, or PMA/ionomycin was found to be blunted within all T cell subpopulations in those with progressive HIV disease compared with long-term nonprogressors and responders. Although alterations in cellular signaling correlated with levels of basal phosphorylation, viral load, and/or expression of programmed death-1, it was the level of basal phosphorylation that appeared to be the factor most dominantly associated with impaired signaling. Notably, provision of effective antiretroviral therapy was associated with a normalization of both basal phosphorylation levels and T cell signaling. These data, in aggregate, suggest that generalized dysfunction of the T cell compartment during progressive HIV disease may be in part dependent upon an increased basal level of phosphorylation, which itself may be due to the heightened state of immune activation found in advanced disease.
PMCID: PMC2648824  PMID: 18453567

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