Comparative analysis of human gut- and blood-derived mononuclear cells: contrasts in function and phenotype.

Introduction: Alterations in the gut immune system have been implicated in various diseases.The challenge of obtaining gut tissues from healthy individuals, commonly performed via surgical explants, has limited the number of studies describing the phenotype and function of gut-derived immune cells in health. Methods: Here, by means of recto-sigmoid colon biopsies obtained during routine care (colon cancer screening in healthy adults), the phenotype and function of immune cells present in the gut were described and compared to those found in blood. Results: The proportion of CD4+, CD8+, MAIT, γδ+ T, and NK cells phenotype, expression of integrins, and ability to produce cytokine in response to stimulation with PMA and ionomycin. T cells in the gut were found to predominantly have a memory phenotype as compared to T cells in blood where a naïve phenotype predominates. Recto-sigmoid mononuclear cells also had higher PD-1 and Ki67 expression. Furthermore, integrin expression and cytokine production varied by cell type and location in blood vs. gut. Discussion: These findings demonstrate the differences in functionality of these cells when compared to their blood counterparts and validate previous studies on phenotype within gut-derived immune cells in humans (where cells have been obtained through surgical means). This study suggests that recto-sigmoid biopsies collected during colonoscopy can be a reliable yet more accessible sampling method for follow up of alterations of gut derived immune cells in clinical settings.

Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.

Background: The differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-ß. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-ß mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear. Methods: Thymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-ß treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation. Results: Despite high expression of CCR5 and CXCR4 by tTregs, FoxP3 + CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-ß1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-ß1. Conclusion: FoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-ß treatment.

Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract.

We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.

Soluble CD127 potentiates IL-7 activity in vivo in healthy mice.

INTRODUCTION: Soluble forms of cytokine receptors can be involved in the endogenous regulation of cytokine activity. Soluble interleukin 7 receptor α (sCD127) naturally binds IL-7, therefore there is interest in its potential application as an immunotherapeutic agent to regulate IL-7. With the hypothesis that sCD127 enhances IL-7 activity, thus promoting T-cell proliferation in vivo, we sought to assess the effect of sCD127, IL-7 or IL-7 + sCD127 treatment on CD4+ and CD8+ T-cells in the blood and spleen of mice. METHODS: Peripheral blood mononuclear cells and splenocytes were prepared, and analyzed for T-cell number, phenotype and proliferation (Ki67+ ) by flow cytometry. RESULTS: IL-7 treatment induced T-cell proliferation, increased T-cell number, and triggered T-cell differentiation each of which was enhanced with the addition of sCD127. IL-7 + sCD127 treatment significantly increased spleen weight over that seen with IL-7 treatment alone. More pronounced proliferation and a greater increase in cell number was observed in CD8+ T-cells relative to the effect on CD4+ T-cells. CONCLUSIONS: These findings suggest that the addition of sCD127 enhances IL-7-mediated T-cell proliferation and suggests a potential therapeutic use for sCD127.

HIV-Infected Macrophages Are Infected and Killed by the Interferon-Sensitive Rhabdovirus MG1.

The use of unique cell surface markers to target and eradicate HIV-infected cells has been a longstanding objective of HIV-1 cure research. This approach, however, overlooks the possibility that intracellular changes present within HIV-infected cells may serve as valuable therapeutic targets. For example, the identification of dysregulated antiviral signaling in cancer has led to the characterization of oncolytic viruses capable of preferentially killing cancer cells. Since impairment of cellular antiviral machinery has been proposed as a mechanism by which HIV-1 evades immune clearance, we hypothesized that HIV-infected macrophages (an important viral reservoir in vivo) would be preferentially killed by the interferon-sensitive oncolytic Maraba virus MG1. We first showed that HIV-infected monocyte-derived macrophages (MDM) were more susceptible to MG1 infection and killing than HIV-uninfected cells. As MG1 is highly sensitive to type I interferons (IFN-I), we then investigated whether we could identify IFN-I signaling differences between HIV-infected and uninfected MDM and found evidence of impaired IFN-α responsiveness within HIV-infected cells. Finally, to assess whether MG1 could target a relevant, primary cell reservoir of HIV-1, we investigated its effects in alveolar macrophages (AM) obtained from effectively treated individuals living with HIV-1. As observed with in vitro-infected MDM, we found that HIV-infected AM were preferentially eliminated by MG1. In summary, the oncolytic rhabdovirus MG1 appears to preferentially target and kill HIV-infected cells via impairment of antiviral signaling pathways and may therefore provide a novel approach to an HIV-1 cure.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) remains a treatable, but incurable, viral infection. The establishment of viral reservoirs containing latently infected cells remains the main obstacle in the search for a cure. Cure research has also focused on only one cellular target of HIV-1 (the CD4+ T cell) while largely overlooking others (such as macrophages) that contribute to HIV-1 persistence. In this study, we address these challenges by describing a potential strategy for the eradication of HIV-infected macrophages. Specifically, we show that an engineered rhabdovirus-initially developed as a cancer therapy-is capable of preferential infection and killing of HIV-infected macrophages, possibly via the same altered antiviral signaling seen in cancer cells. As this rhabdovirus is currently being explored in phase I/II clinical trials, there is potential for this approach to be readily adapted for use within the HIV-1 cure field.

IL-7 induces sCD127 release and mCD127 downregulation in human CD8+ T cells by distinct yet overlapping mechanisms, both of which are impaired in HIV infection.

The IL-7 receptor specific α chain, CD127, can be expressed both as a membrane-associated (mCD127) and a soluble form (sCD127), however, the mechanisms involved in their regulation remain to be defined. We first demonstrated in primary human CD8+ T cells that IL-7-induced downregulation of mCD127 expression is dependent on JAK and PI3K signaling, whereas IL-7-induced sCD127 release is also mediated by STAT5. Following stimulation with IL-7, expression of alternatively spliced variants of the CD127 gene, sCD127 mRNA, is reduced, but to a lesser degree than the full-length gene. Evaluation of the role of proteases revealed that MMP-9 was involved in sCD127 release, without affecting the expression of mCD127, suggesting it does not induce direct shedding from the cell surface. Since defects in the IL-7/CD127 pathway occur in various diseases, including HIV, we evaluated CD8+ T cells derived from HAART-treated HIV-infected individuals and found that IL-7-induced (1) downregulation of mCD127, (2) release of sCD127, and (3) expression of the sCD127 mRNA were all impaired. Expression of mCD127 and sCD127 is, therefore, regulated by distinct, but overlapping, mechanisms and their impairment in HIV infection contributes to our understanding of the CD8+ T cell dysfunction that persists despite effective antiretroviral therapy.

IL-7-induced proliferation of peripheral Th17 cells is impaired in HAART-controlled HIV infection.

OBJECTIVES: Th17 cells are key regulators of functional immunity in mucosal tissues, including the gut-associated lymphoid tissue (GALT), an important site of immune impairment in HIV infection. During HIV infection, Th17 cells are lost in large numbers from the GALT. Despite the recovery of peripheral CD4 T cells that accompanies suppression of viral replication with HAART, Th17 cells in GALT are not completely restored. IL-7 is essential for the survival and proliferation of T cells, but its signaling through its receptor IL-7Rα (CD127), is impaired in CD8 T cells and thymocytes during HIV infection. We set out to determine if decreased CD127 expression or impaired CD127 signaling may be the cause of Th17 impairment in HAART-controlled HIV infection. DESIGN: Healthy and HIV donors on HAART were selected for this study of Th17 cell function in HIV. METHODS: Peripheral CD4 T cells and Th17 cells were isolated using magnetic beads, then stimulated with IL-7. CD127 expression and the phosphorylation of signaling molecules was determined using flow cytometry. Proliferation was determined with a CFSE dilution assay. RESULTS: CD127 was not decreased on Th17 cells from HAART-controlled HIV individuals, in fact, the percentage of Th17 cells that express CD127 was increased in treated HIV individuals. Furthermore, Th17 cells from HAART-controlled individuals, have normal IL-7-induced STAT5 and Bcl-2 responses, but vastly decreased proliferative responses. CONCLUSION: This reduced IL-7 responsiveness may explain the lack of Th17 cell recovery and ongoing systemic immune activation that persists despite well treated HIV infection.

Medical Assistance in Death as a Unique Opportunity to Advance Human Immunodeficiency Virus Cure Research.

The inability to sample deep-tissue reservoirs in individuals living with human immunodeficiency virus (HIV) has greatly hindered accurate estimates of viral reservoir size and distribution. Animal models and collection of tissues during autopsies of HIV-positive individuals are 2 proposed solutions to this problem. Each, however, has its limitations. In this Viewpoint, we argue that tissue donation following medical assistance in death (MAiD) will form an invaluable resource for the characterization of the viral reservoir in the context of current HIV cure research. In support, we discuss a recent instance in which an individual living with HIV chose to donate their body/tissues to HIV research prior to undergoing MAiD at our institution. Going forward, we hope this will help provide support to individuals in their decisions around tissue donation following MAiD, while highlighting how healthcare providers, by complying with such wishes, can affect patient satisfaction in the last days of life.

The Oncolytic Virus MG1 Targets and Eliminates Cells Latently Infected With HIV-1: Implications for an HIV Cure.

Cells latently infected with human immunodeficiency virus (HIV) evade immune- and drug-mediated clearance. These cells harbor intracellular signaling defects, including impairment of the antiviral type I interferon response. Such defects have also been observed in several cancers and have been exploited for the development of therapeutic oncolytic viruses, including the recombinant Maraba virus (MG1). We therefore hypothesized that MG1 would infect and eliminate cells latently infected with HIV-1, while sparing healthy uninfected cells. Preferential infection and elimination by MG1 was first demonstrated in cell lines latently infected with HIV-1. Following this, a reduction in HIV-1 DNA and inducible HIV-1 replication was observed following MG1 infection of latently infected, resting CD4+ T cells generated using an in vitro model of latency. Last, MG1 infection resulted in a reduction in HIV-1 DNA and inducible HIV-1 replication in memory CD4+ T cells isolated from effectively treated, HIV-1-infected individuals. Our results therefore highlight a novel approach to eliminate the latent HIV-1 reservoir.

Generalized Liver- and Blood-Derived CD8+ T-Cell Impairment in Response to Cytokines in Chronic Hepatitis C Virus Infection.

Generalized CD8+ T-cell impairment in chronic hepatitis C virus (HCV) infection and the contribution of liver-infiltrating CD8+ T-cells to the immunopathogenesis of this infection remain poorly understood. It is hypothesized that this impairment is partially due to reduced CD8+ T-cell activity in response to cytokines such as IL-7, particularly within the liver. To investigate this, the phenotype and cytokine responsiveness of blood- and liver-derived CD8+ T-cells from healthy controls and individuals with HCV infection were compared. In blood, IL-7 receptor α (CD127) expression on bulk CD8+ T-cells in HCV infection was no different than controls yet was lower on central memory T-cells, and there were fewer naïve cells. IL-7-induced signalling through phosphorylated STAT5 was lower in HCV infection than in controls, and differed between CD8+ T-cell subsets. Production of Bcl-2 following IL-7 stimulation was also lower in HCV infection and inversely related to the degree of liver fibrosis. In liver-derived CD8+ T-cells, STAT5 activation could not be increased with cytokine stimulation and basal Bcl-2 levels of liver-derived CD8+ T-cells were lower than blood-derived counterparts in HCV infection. Therefore, generalized CD8+ T-cell impairment in HCV infection is characterized, in part, by impaired IL-7-mediated signalling and survival, independent of CD127 expression. This impairment is more pronounced in the liver and may be associated with an increased potential for apoptosis. This generalized CD8+ T-cell impairment represents an important immune dysfunction in chronic HCV infection that may alter patient health.