Kynurenine 3-Monooxygenase Inhibition during Acute Simian Immunodeficiency Virus Infection Lowers PD-1 Expression and Improves Post-Combination Antiretroviral Therapy CD4+ T Cell Counts and Body Weight.

The kynurenine pathway (KP) is a key regulator of many important physiological processes and plays a harmful role in cancer, many neurologic conditions, and chronic viral infections. In HIV infection, KP activity is consistently associated with reduced CD4 T cell counts and elevated levels of T cell activation and viral load; it also independently predicts mortality and morbidity from non-AIDS events. Kynurenine 3-monooxygenase (KMO) is a therapeutically important target in the KP. Using the nonhuman primate model of SIV infection in rhesus macaques, we investigated whether KMO inhibition could slow the course of disease progression. We used a KMO inhibitor, CHDI-340246, to perturb the KP during early acute infection and followed the animals for 1 y to assess clinical outcomes and immune phenotype and function during pre-combination antiretroviral therapy acute infection and combination antiretroviral therapy-treated chronic infection. Inhibition of KMO in acute SIV infection disrupted the KP and prevented SIV-induced increases in downstream metabolites, improving clinical outcome as measured by both increased CD4+ T cell counts and body weight. KMO inhibition increased naive T cell frequency and lowered PD-1 expression in naive and memory T cell subsets. Importantly, early PD-1 expression during acute SIV infection predicted clinical outcomes of body weight and CD4+ T cell counts. Our data indicate that KMO inhibition in early acute SIV infection provides clinical benefit and suggest a rationale for testing KMO inhibition as an adjunctive treatment in SIV/HIV infection to slow the progression of the disease and improve immune reconstitution.

Defining total-body AIDS-virus burden with implications for curative strategies.

In the quest for a functional cure or the eradication of HIV infection, it is necessary to know the sizes of the reservoirs from which infection rebounds after treatment interruption. Thus, we quantified SIV and HIV tissue burdens in tissues of infected nonhuman primates and lymphoid tissue (LT) biopsies from infected humans. Before antiretroviral therapy (ART), LTs contained >98% of the SIV RNA+ and DNA+ cells. With ART, the numbers of virus (v) RNA+ cells substantially decreased but remained detectable, and their persistence was associated with relatively lower drug concentrations in LT than in peripheral blood. Prolonged ART also decreased the levels of SIV- and HIV-DNA+ cells, but the estimated size of the residual tissue burden of 108 vDNA+ cells potentially containing replication-competent proviruses, along with evidence of continuing virus production in LT despite ART, indicated two important sources for rebound following treatment interruption. The large sizes of these tissue reservoirs underscore challenges in developing 'HIV cure' strategies targeting multiple sources of virus production.

Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing.

UNLABELLED: Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4(+) T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs. IMPORTANCE: As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4(+) T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4(+) T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4(+) T cells will likely dictate different approaches in order to achieve their elimination. For CD4(+) T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8(+) T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

Exposure to SIV in utero results in reduced viral loads and altered responsiveness to postnatal challenge.

HIV disease progression appears to be driven by increased immune activation. Given observations that fetal exposure to infectious pathogens in utero can result in reduced immune responses, or tolerance, to those pathogens postnatally, we hypothesized that fetal exposure to HIV may render the fetus tolerant to the virus, thus reducing damage caused by immune activation during infection later in life. To test this hypothesis, fetal rhesus macaques (Macaca mulatta) were injected with the attenuated virus SIVmac1A11 in utero and challenged with pathogenic SIVmac239 1 year after birth. SIVmac1A11-injected animals had significantly reduced plasma RNA viral loads (P < 0.02) up to 35 weeks after infection. Generalized estimating equations analysis was performed to identify immunologic and clinical measurements associated with plasma RNA viral load. A positive association with plasma RNA viral load was observed with the proportion of CD8(+) T cells expressing the transcription factor, FoxP3, and the proportion of CD4(+) T cells producing the lymphoproliferative cytokine, IL-2. In contrast, an inverse relationship was found with the frequencies of circulating CD4(+) and CD8(+) T cells displaying intermediate expression of the proliferation marker, Ki-67. Animals exposed to simian immunodeficiency virus (SIV) in utero appeared to have enhanced SIV-specific immune responses, a lower proportion of CD8(+) T cells expressing the exhaustion marker PD-1, and more circulating TH17 cells than controls. Although the development of tolerance was not demonstrated, these data suggest that rhesus monkeys exposed to SIVmac1A11 in utero had distinct immune responses associated with the control of viral replication after postnatal challenge.

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 midgestation 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.

Higher CD27+CD8+ T cells percentages during suppressive antiretroviral therapy predict greater subsequent CD4+ T cell recovery in treated HIV infection.

HIV-mediated immune dysfunction may influence CD4(+) T cell recovery during suppressive antiretroviral therapy (ART). We analyzed cellular biomarkers of immunological inflammation, maturation, and senescence in HIV-infected subjects on early suppressive ART. We performed longitudinal analyses of peripheral immunological biomarkers of subjects on suppressive ART (n = 24) from early treatment (median 6.4 months, interquartile range [IQR] 4.8-13.9 months) to 1-2 years of follow-up (median 19.8 months, IQR 18.3-24.6 months). We performed multivariate regression to determine which biomarkers were associated with and/or predictive of CD4(+) T cell recovery. After adjusting for the pre-ART CD4(+) T cell count, age, proximal CD4(+) T cell count, and length of ART medication, the percentage of CD27(+)CD8(+) T cells remained significantly associated with the CD4(+) T cell recovery rate (ß = 0.092 cells/ul/month, P = 0.028). In HIV-infected subjects starting suppressive ART, patients with the highest percentage of CD8(+) T cells expressing CD27 had the greatest rate of CD4(+) T cell recovery.

Molecular and cellular basis of T cell lineage commitment.

The thymus forms as an alymphoid thymic primordium with T cell differentiation requiring the seeding of this anlage. This review will focus on the characteristics of the hematopoietic progenitors which colonize the thymus and their subsequent commitment/differentiation, both in mice and men. Within the thymus, the interplay between Notch1 and IL-7 signals is crucial for the orchestration of T cell development, but the precise requirements for these factors in murine and human thympoeisis are not synonymous. Recent advances in our understanding of the mechanisms regulating precursor entry and their maintenance in the thymus will also be presented.

In vivo and ex vivo gene transfer in thymocytes and thymocyte precursors.

The thymus provides a specialized environment allowing the differentiation of T lymphocytes from bone marrow-derived progenitor cells. We and others have demonstrated that gene transfer into distinct thymocyte populations can be obtained, both in vivo and ex vivo, using lentiviral vectors. Here, we describe techniques for intrathymic lentiviral transduction in mice, using a surgical approach wherein the thoracic cavity is exposed as well as a significantly less invasive strategy wherein virions are directly injected through the skin. Moreover, thymocyte differentiation from murine and human progenitors is now feasible in vitro, under conditions wherein the Notch and IL-7 signaling pathways are activated. We describe methods allowing transduction of murine and human progenitors and their subsequent differentiation into more mature thymocytes. Conditions for lentiviral gene transfer into more differentiated human thymocyte subsets are also presented. Optimization of technologies for HIV-based gene transfer into murine and human thymocyte progenitors will advance strategies aimed at modulating T-cell differentiation and function in-vivo; approaches potentially targeting patients with genetic and acquired immunodeficiencies as well as immune-sensitive tumors. Furthermore, this technology will foster the progression of basic research aimed at elucidating molecular aspects of T-cell differentiation in mice and humans.

Lentiviral transduction of immune cells.

Gene transfer into mammalian cells has been of crucial importance for studies determining the role of specific genes in the differentiation and cell fate of various hematopoietic lineages. Until recently, the majority of these studies were performed in transformed cell lines due to difficulties in achieving levels of transfection of greater than 1-3% in primary hematopoietic cells. Vectors based on retrovirus and lentivirus backbones have revolutionized our ability to transfer genes into primary hematopoietic cells. These vectors have allowed extensive ex vivo and in vivo studies following introduction of a gene of interest and have been used clinically in individuals suffering from cancers, infections, and genetic diseases. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells (>80%) that can then be further differentiated into immune lineage cells including T, B, dendritic, or natural killer cells. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Here, we discuss optimization of technologies for human immunodeficiency virus (HIV)-based gene transfer into murine and human progenitor and immune cell lineages.

Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells.

BACKGROUND: We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. RESULTS: As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. CONCLUSION: Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes.