Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry.

CONTEXT.­: Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal towards standardization of the Tregs assay. OBJECTIVE.­: To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets. DESIGN.­: The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of CD4+CD25+CD127low/- Tregs and CD4+CD25+Foxp3+ Tregs was evaluated. RESULTS.­: The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with R2 ≥0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the CD3+CD4+ gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of CD4+CD25+CD127low/- Tregs was closely correlated with CD4+CD25+Foxp3+ Tregs. CONCLUSIONS.­: This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice.

Altered lipid metabolites accelerate early dysfunction of T cells in HIV-infected rapid progressors by impairing mitochondrial function.

The complex mechanism of immune-system damage in HIV infection is incompletely understood. HIV-infected "rapid progressors" (RPs) have severe damage to the immune system early in HIV infection, which provides a "magnified" opportunity to study the interaction between HIV and the immune system. In this study, forty-four early HIV-infected patients (documented HIV acquisition within the previous 6 months) were enrolled. By study the plasma of 23 RPs (CD4+ T-cell count < 350 cells/µl within 1 year of infection) and 21 "normal progressors" (NPs; CD4+ T-cell count > 500 cells/µl after 1 year of infection), eleven lipid metabolites were identified that could distinguish most of the RPs from NPs using an unsupervised clustering method. Among them, the long chain fatty acid eicosenoate significantly inhibited the proliferation and secretion of cytokines and induced TIM-3 expression in CD4+ and CD8+ T cells. Eicosenoate also increased levels of reactive oxygen species (ROS) and decreased oxygen consumption rate (OCR) and mitochondrial mass in T cells, indicating impairment in mitochondrial function. In addition, we found that eicosenoate induced p53 expression in T cells, and inhibition of p53 effectively decreased mitochondrial ROS in T cells. More importantly, treatment of T cells with the mitochondrial-targeting antioxidant mito-TEMPO restored eicosenoate-induced T-cell functional impairment. These data suggest that the lipid metabolite eicosenoate inhibits immune T-cell function by increasing mitochondrial ROS by inducing p53 transcription. Our results provide a new mechanism of metabolite regulation of effector T-cell function and provides a potential therapeutic target for restoring T-cell function during HIV infection.

Alterations in levels of cytokine following treatment to predict outcome of sepsis: A meta-analysis.

BACKGROUND: The mortality rate of patients with sepsis has been increasing in recent years. Alterations of biomarkers levels during treatment are important in evaluating treatment efficacy and predicting outcomes in sepsis. This meta-analysis investigated the relationship between changes in cytokine levels after treatment compared with those on hospital admission, and their relationship with the prognosis of patients with sepsis. METHODS: From conception until August 4, 2021, a complete literature search of the PubMed, Web of Science, and Cochrane Library electronic databases was done. Observational studies where the outcomes of sepsis patients were divided into non-survivors and survivors and which reported cytokine levels at least before treatment in ICU were included in the current study. Standardized mean difference (SMD) with 95% confidence intervals (CI) values from individual studies were pooled using a random-effects model. Quality assessment, subgroup analysis, publication bias, and sensitivity analyses were all carried out. RESULTS: A total of 2570 patients with sepsis from 25 eligible studies were included, and 14 of them measured the cytokine levels before and after treatment in ICU. Among IL-6, TNF-α, IL-1ß and IL-10 levels, those of IL-6 were significantly lower after treatment in ICU than at baseline in patients with sepsis in the survival group (SMD = -0.69, P < 0.0001), but were comparable in the non-survival group (SMD = -0.99, P = 0.0575). Similarly, post-treatment TNF-α levels were significantly lower than those at baseline only in patients with sepsis in the survival group (SMD = -0.44, P < 0.0001), but not in the non-survival group (SMD =-0.17, P = 0.0842). CONCLUSION: This meta-analysis shows that reduced IL-6 and TNF-α levels after sepsis treatment in ICU may be indicators of better prognosis and survival of patients with sepsis.

Immunomodulatory PEG-CRGD Hydrogels Promote Chondrogenic Differentiation of PBMSCs.

Cartilage damage is a common injury. Currently, tissue engineering scaffolds with composite seed cells have emerged as a promising approach for cartilage repair. Polyethylene glycol (PEG) hydrogels are attractive tissue engineering scaffold materials as they have high water absorption capacity as well as nontoxic and nutrient transport properties. However, PEG is fundamentally bio-inert and lacks intrinsic cell adhesion capability, which is critical for the maintenance of cell function. Cell adhesion peptides are usually added to improve the cell adhesion capability of PEG-based hydrogels. The suitable cell adhesion peptide can not only improve cell adhesion capability, but also promote chondrogenesis and regulate the immune microenvironment. To improve the interactions between cells and PEG hydrogels, we designed cysteine-arginine-glycine-aspartic acid (CRGD), a cell adhesion peptide covalently cross-linked with PEG hydrogels by a Michael addition reaction, and explored the tissue-engineering hydrogels with immunomodulatory effects and promoted chondrogenic differentiation of mesenchymal stem cells (MSCs). The results indicated that CRGD improved the interaction between peripheral blood mesenchymal stem cells (PBMSCs) and PEG hydrogels. PEG hydrogels modified with 1 mM CRGD had the optimal capacity to promote chondrogenic differentiation, and CRGD could induce macrophage polarization towards the M2 phenotype to promote tissue regeneration and repair. PEG-CRGD hydrogels combined with PBMSCs have the potential to be suitable scaffolds for cartilage tissue engineering.

Downregulation of TCF1 in HIV Infection Impairs T-cell Proliferative Capacity by Disrupting Mitochondrial Function.

Background: Despite the benefits of antiretroviral therapy (ART) for people with HIV, T-cell dysfunction cannot be fully restored. Metabolic dysregulation is associated with dysfunction of HIV-1-specific T-cells. Exploration of the factors regulating metabolic fitness can help reverse T-cell dysfunction and provide new insights into the underlying mechanism. Methods: In this study, HIV-infected individuals and HIV-negative control individuals (NCs) were enrolled. T-cell factor (TCF)1 expression in cells was determined by quantitative reverse-transcriptase polymerase chain reaction and flow cytometry. Relevant microarray data from the GEO database were analyzed to explore the underlying mechanism. The effects of TCF1 on T-cell function and metabolic function were assessed in vitro. Results: TCF7 mRNA expression in peripheral blood mononuclear cells was downregulated in rapid progressors compared with long-term non-progressors individuals and NCs. TCF1 expression on CD4+ and CD8+ T-cells was downregulated in treatment-naïve HIV-infected individuals compared with NCs. Interleukin (IL)2 production and proliferative capacity were impaired in TCF1 knockdown T-cells. Moreover, glycolytic capacity and mitochondrial respiratory function were decreased in TCF1 knockdown T-cells, and depolarized mitochondria were increased in TCF1 knockdown T-cells. Conclusion: Downregulation of TCF1 in HIV infection impairs T-cell proliferative capacity by disrupting mitochondrial function. These findings highlight the metabolic regulation as a pivotal mechanism of TCF1 in the regulation of T-cell dysfunction.

The development of autoverification system of lymphocyte subset assays on the flow cytometry platform.

OBJECTIVES: Peripheral blood lymphocyte subsets are important parameters for monitoring immune status; however, lymphocyte subset detection is time-consuming and error-prone. This study aimed to explore a highly efficient and clinically useful autoverification system for lymphocyte subset assays performed on the flow cytometry platform. METHODS: A total of 94,402 lymphocyte subset test results were collected. To establish the limited-range rules, 80,427 results were first used (69,135 T lymphocyte subset tests and 11,292 NK, B, T lymphocyte tests), of which 15,000 T lymphocyte subset tests from human immunodeficiency virus (HIV) infected patients were used to set customized limited-range rules for HIV infected patients. Subsequently, 13,975 results were used for historical data validation and online test validation. RESULTS: Three key autoverification rules were established, including limited-range, delta-check, and logical rules. Guidelines for addressing the issues that trigger these rules were summarized. The historical data during the validation phase showed that the total autoverification passing rate of lymphocyte subset assays was 69.65% (6,941/9,966), with a 67.93% (5,268/7,755) passing rate for T lymphocyte subset tests and 75.67% (1,673/2,211) for NK, B, T lymphocyte tests. For online test validation, the total autoverification passing rate was 75.26% (3,017/4,009), with 73.23% (2,191/2,992) for the T lymphocyte subset test and 81.22% (826/1,017) for the NK, B, T lymphocyte test. The turnaround time (TAT) was reduced from 228 to 167 min using the autoverification system. CONCLUSIONS: The autoverification system based on the laboratory information system for lymphocyte subset assays reduced TAT and the number of error reports and helped in the identification of abnormal cell populations that may offer clues for clinical interventions.

Plasma MicroRNA Signature Panel Predicts the Immune Response After Antiretroviral Therapy in HIV-Infected Patients.

Background: Approximately 10-40% of people with human immunodeficiency virus (HIV) infection are unable to obtain successful improvements in immune function after antiretroviral therapy (ART). These patients are at greater risk of developing non-acquired immunodeficiency syndrome (AIDS)-related conditions, with the accompanying increased morbidity and mortality. Discovering predictive biomarkers can help to identify patients with a poor immune response earlier and provide new insights into the mechanisms of this condition. Methods: A total of 307 people with HIV were enrolled, including 110 immune non-responders (INRs) and 197 immune responders (IRs). Plasma samples were taken before ART, and quantities of plasma microRNAs (miRNAs) were determined using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Candidate biomarkers were established through four phases: discovery, training, validation, and blinded test. Binary logistic regression was used to analyze the combined predictive capacity of the identified miRNAs. The effect of one miRNA, miR-16-5p, on T cell function was assessed in vitro. Results: Expression of five miRNAs (miR-580, miR-627, miR-138-5p, miR-16-5p, and miR-323-3p) was upregulated in the plasma of INRs compared with that in IRs. Expression of these miRNAs was negatively correlated with both CD4+ T cell counts and the increase in the proportion of CD4+ T cells after one year of ART. These five miRNAs were combined in a predictive model, which could effectively identify INRs or IRs. Furthermore, we found that miR-16-5p inhibits CD4+ T cell proliferation by regulating calcium flux. Conclusion: We established a five-miRNA panel in plasma that accurately predicts poor immune response after ART, which could inform strategies to reduce the incidence of this phenomenon and improve the clinical management of these patients.

Prevention and control of HIV/AIDS in China: lessons from the past three decades.

ABSTRACT: In the past 37 years, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) has undergone various major transmission routes in China, with the world most complex co-circulating HIV-1 subtypes, even the prevalence is still low. In response to the first epidemic outbreak of HIV in injecting drug users and the second one by illegal commercial blood collection, China issued the Anti-Drug Law and launched the Blood Donation Act and nationwide nucleic acid testing, which has avoided 98,232 to 211,200 estimated infections and almost ended the blood product-related infection. China has been providing free antiretroviral therapy (ART) since 2003, which covered >80% of the identified patients and achieved a viral suppression rate of 91%. To bend the curve of increasing the disease burden of HIV and finally end the epidemic, China should consider constraining HIV spread through sexual transmission, narrowing the gaps in identifying HIV cases, and the long-term effectiveness and safety of ART in the future.

Scaffold-Based Tissue Engineering Strategies for Osteochondral Repair.

Over centuries, several advances have been made in osteochondral (OC) tissue engineering to regenerate more biomimetic tissue. As an essential component of tissue engineering, scaffolds provide structural and functional support for cell growth and differentiation. Numerous scaffold types, such as porous, hydrogel, fibrous, microsphere, metal, composite and decellularized matrix, have been reported and evaluated for OC tissue regeneration in vitro and in vivo, with respective advantages and disadvantages. Unfortunately, due to the inherent complexity of organizational structure and the objective limitations of manufacturing technologies and biomaterials, we have not yet achieved stable and satisfactory effects of OC defects repair. In this review, we summarize the complicated gradients of natural OC tissue and then discuss various osteochondral tissue engineering strategies, focusing on scaffold design with abundant cell resources, material types, fabrication techniques and functional properties.

Function and Mechanism of RGD in Bone and Cartilage Tissue Engineering.

Bone and cartilage injury is common, tissue engineered scaffolds are potential means to repair. Because most of the scaffold materials used in bone and cartilage tissue engineering are bio-inert, it is necessary to increase the cellular adhesion ability of during tissue engineering reconstruction. The Arginine - Glycine - Aspartic acid (Arg-Gly-Asp, RGD) peptide family is considered as a specific recognition site for the integrin receptors. Integrin receptors are key regulators of cell-cell and cell-extracellular microenvironment communication. Therefore, the RGD polypeptide families are considered as suitable candidates for treatment of a variety of diseases and for the regeneration of various tissues and organs. Many scaffold material for tissue engineering and has been approved by US Food and Drug Administration (FDA) for human using. The application of RGD peptides in bone and cartilage tissue engineering was reported seldom. Only a few reviews have summarized the applications of RGD peptide with alloy, bone cements, and PCL in bone tissue engineering. Herein, we summarize the application progress of RGD in bone and cartilage tissue engineering, discuss the effects of structure, sequence, concentration, mechanical stimulation, physicochemical stimulation, and time stimulation of RGD peptide on cells differentiation, and introduce the mechanism of RGD peptide through integrin in the field of bone and cartilage tissue engineering.

Corrigendum: Increased Expression of sST2 in Early HIV Infected Patients Attenuated the IL-33 Induced T Cell Responses.

[This corrects the article DOI: 10.3389/fimmu.2018.02850.].

CD4+CD38+ central memory T cells contribute to HIV persistence in HIV-infected individuals on long-term ART.

BACKGROUND: Despite the effective antiretroviral treatment (ART) of HIV-infected individuals, HIV persists in a small pool. Central memory CD4+ T cells (Tcm) make a major contribution to HIV persistence. We found that unlike HLA-DR, CD38 is highly expressed on the Tcm of HIV-infected subjects receiving ART for > 5 years. It has been reported that the half-life of total and episomal HIV DNA in the CD4+CD38+ T cell subset, exhibits lower decay rates at 12 weeks of ART. Whether CD38 contributes to HIV latency in HIV-infected individuals receiving long-term ART is yet to be addressed. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of HIV-infected subjects receiving suppressive ART. The immunophenotyping, proliferation and apoptosis of CD4+ T cell subpopulations were detected by flow cytometry, and the level of CD38 mRNA and total HIV DNA were measured using real-time PCR and digital droplet PCR, respectively. A negative binomial regression model was used to determine the correlation between CD4+CD38+ Tcm and total HIV DNA in CD4+ T cells. RESULTS: CD38 was highly expressed on CD4+ Tcm cells from HIV infected individuals on long-term ART. Comparing with HLA-DR-Tcm and CD4+HLA-DR+ T cells, CD4+CD38+ Tcm cells displayed lower levels of activation (CD25 and CD69) and higher levels of CD127 expression. The proportion of CD38+ Tcm, but not CD38- Tcm cells can predict the total HIV DNA in the CD4+ T cells and the CD38+ Tcm subset harbored higher total HIV DNA copy numbers than the CD38- Tcm subset. After transfected with CD38 si-RNA in CD4+ T cells, the proliferation of CD4+ T cells was inhibited. CONCLUSION: The current date indicates that CD4+CD38+ Tcm cells contribute to HIV persistence in HIV-infected individuals on long-term ART. Our study provides a potential target to resolve HIV persistence.

Transcriptional insights into the CD8+ T cell response in mono-HIV and HCV infection.

BACKGROUND: Disease progression in the absence of therapy varies significantly in mono-HIV and HCV infected individuals. Virus-specific CD8+ T cells play an important role in restricting lentiviral replication and determining the rate of disease progression during HIV and HCV mono- and co-infection. Thus, understanding the similarities in the characteristics of CD8+ T cells in mono-HIV and HCV infection at the transcriptomic level contributes to the development of antiviral therapy. In this study, a meta-analysis of CD8+ T cell gene expression profiles derived from mono-HIV and HCV infected individuals at different stages of disease progression, was conducted to understand the common changes experienced by CD8+ T cells. METHODS: Five microarray datasets, reporting CD8+ T cell mRNA expression of the mono-HIV and HCV infected patients, were retrieved from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified via integrative meta-analysis of expression data (INMEX) program. Network analysis methods were used to assess protein-protein interaction (PPI) networks, Gene Ontology (GO) terms and pathway enrichment for DEGs. MirDIP and miRDB online prediction tools were used to predict potential microRNAs (miRNAs) targeting hub genes. RESULTS: First, we identified 625 and 154 DEGs in the CD8+ T cells originating from mono-HIV and HCV chronic progressor patients, respectively, compared to healthy individuals. Among them, interferon-stimulated genes (ISGs) including ISG15, IFIT3, ILI44L, CXCL8, FPR1 and TLR2, were upregulated after mono-HIV and HCV infection. Pathway enrichment analysis of DEGs showed that the "cytokine-cytokine receptor interaction" and "NF-kappa B" signaling pathways were upregulated after mono-HIV and HCV infection. In addition, we identified 92 and 50 DEGs in the CD8+ T cells of HIV non-progressor and HCV resolver patients, respectively, compared with corresponding chronic progressors. We observed attenuated mitosis and reduced ISG expression in HIV non-progressors and HCV resolvers compared with the corresponding chronic progressors. Finally, we identified miRNA-143-3p, predicted to target both IFIT3 in HIV and STAT5A in HCV infection. CONCLUSIONS: We identified DEGs and transcriptional patterns in mono-HIV and HCV infected individuals at different stages of disease progression and identified miRNA-143-3p with potential to intervene disease progression, which provides a new strategy for developing targeted therapies.

Decreased CD4+CD8low T cells in early HIV infection are associated with rapid disease progression.

BACKGROUND: HIV rapid progressors (RPs) present with a rapid decline of CD4+ T cells within a few years of infection. Determining the underlying mechanisms throughout this decline is important to identify prognostic biomarkers and intervention strategies. Determining the numbers of CD4+ and CD8+ T cells is essential for monitoring the immune status of HIV infected patients. There are additional kinds of cell subtypes in T cells, but their relationship to the rapid progression of HIV disease is not well defined. METHODS: Nineteen RPs and twenty-one chronic progressors (CPs) were enrolled in this study. Based on the intensity of CD4 and CD8 expression, different T cell subtypes were identified, including CD4+CD8+T cells, CD4-CD8- T cells, CD4+CD8low T cells and CD4-CD8low T cells. Alterations in these T cell subtypes in early HIV infection (within 120 days of infection) between RPs and CPs were measured, and the relationships between these subtypes and HIV disease progression were investigated. In addition, expression of IFN-γ in T cell subtypes after PMA stimulation was analyzed by flow cytometry. RESULTS: We found that during early HIV infection, CD4+CD8low T cells both significantly decreased in numbers and percentages in RPs compared to CPs. Furthermore, baseline CD4+CD8low T cells positively correlated not only with baseline CD4+T cells but also with CD4+T cells 12 months after infection. Moreover, survival analysis indicated that low levels of baseline CD4+CD8low T cells significantly accelerated the decline in CD4+ T cells as well as increased viral loads. CD4+CD8low T cells secreted significantly more IFN-γ after PMA stimulation compared to CD4+CD8-T cells and CD4-CD8+T cells, which may be beneficial for the prevention of disease progression. CONCLUSIONS: Our results identified that in early stage HIV-1 infection, a subtype of T cells, CD4+CD8low, are associated with subsequent disease progression.

Reduced eIF3d accelerates HIV disease progression by attenuating CD8+ T cell function.

BACKGROUND: In human immunodeficiency virus (HIV) infection, 10-15% of individuals exhibit a rapid decline in CD4+ T cells and become rapid progressors (RPs). Overall, understanding the factors affecting rapid disease progression in early HIV infection (EHI) can aid in treatment initiation. Recent studies show that eIF3s, classic scaffold proteins during the translation initiation process, can directly promote or inhibit the translation of mRNA, therefore participating in the regulation of cell function. However, to our knowledge, it has not been addressed whether eIF3s are involved in the diverse prognosis of HIV infection. METHODS: Expression of eIF3s in primary cells from early or chronic HIV-infected patients was detected by real-time PCR. To investigate the potential mechanisms of eIF3d in the regulation of CD8+ T cell function, complete transcriptomes of eIF3d-inhibited Jurkat T cells were sequenced by RNA sequencing (RNA-Seq). Additionally, to examine the effect of eIF3d on CD8+ T cell function, eIF3d expression was inhibited alone or in combination with SOCS-7 knockdown by siRNA in isolated CD8+ T cells. CD8+ T cell proliferation, IFN-r secretion and apoptosis were detected by flow cytometry. Moreover, the effect of eIF3d on HIV replication was evaluated in Jurkat cells, peripheral blood mononuclear cells (PBMCs) and CD4+ T cells with eIF3d knockdown using a pNL4-3 pseudotyped virus. RESULTS: At approximately 100 days of infection, only eIF3d was markedly decreased in RPs compared with chronic progressors (CPs). Expression of eIF3d correlated significantly with disease progression in EHI. Based on in vitro analyses, reduced eIF3d expression led to decreased proliferation and IFN-γ secretion and increased apoptosis in CD8+ T cells. Inhibited expression of eIF3d caused enhanced expression of SOCS-7, and inhibiting SOCS-7 expression by siRNA rescued the attenuated CD8+ T cell function caused by eIF3d. Finally, when eIF3d was inhibited in Jurkat cells, PBMCs and CD4+ T cells, pNL4-3-VSV-G virus replication was enhanced. CONCLUSIONS: The current data highlight the importance of eIF3d in HIV infection by inhibiting CD8+ T cell function and promoting viral replication. Our study provides potential targets for improved immune intervention.

Increased Expression of sST2 in Early HIV Infected Patients Attenuated the IL-33 Induced T Cell Responses.

T cell responses were less functional and persisted in an exhausted state in chronic HIV infection. Even in early phase of HIV infection, the dysfunction of HIV-specific T cells can be observed in rapid progressors, but the underlying mechanisms are not fully understood. Cytokines play a central role in regulating T cell function. In this study, we sought to elucidate whether IL-33/ST2 axis plays roles in the regulation of T cell function in HIV infection. We found that the level of IL-33 was upregulated in early HIV-infected patients compared with that in healthy controls and has a trend associated with disease progression. In vitro study shows that IL-33 promotes the expression of IFN-γ by Gag stimulated CD4+ and CD8+T cells from HIV-infected patients to a certain extent. However, soluble ST2 (sST2), a decoy receptor of IL-33, was also increased in early HIV infected patients, especially in those with progressive infection. We found that anti-ST2 antibodies attenuated the effect of IL-33 to CD4+ and CD8+T cells. Our data indicates that elevated expression of IL-33 in early HIV infection has the potential to enhance the function of T cells, but the upregulated sST2 weakens the activity of IL-33, which may indirectly contribute to the dysfunction of T cells and rapid disease progression. This data broadens the understanding of HIV pathogenesis and provides critical information for HIV intervention.

Elevated Expression of miR-19b Enhances CD8+ T Cell Function by Targeting PTEN in HIV Infected Long Term Non-progressors With Sustained Viral Suppression.

Human immunodeficiency virus (HIV)-infected long-term non-progressors (LTNPs) are of particular importance because of their unique disease progression characteristics. Defined by the maintenance of normal CD4+T cells after more than 8 years of infection, these LTNPs are heterogeneous. Some LTNPs exhibit ongoing viral production, while others do not and are able to control viral production. The underlying basis for this heterogeneity has not been clearly elucidated. In this study, the miRNA expression profiles of LTNPs were assessed. The levels of microRNA-19b (miR-19b) were found to be significantly increased in peripheral blood mononuclear cells of LTNPs with lower rather than higher viral load. We made clear that miR-19b may regulate CD8+T cell functions in HIV infection, which has not been addressed before. Overexpression of miR-19b promoted CD8+T cell proliferation, as well as interferon-γ and granzyme B expression, while inhibiting CD8+T cells apoptosis induced by anti-CD3/CD28 stimulation. The target of miR-19b was found to be the "phosphatase and tensin homolog", which regulates CD8+T cells function during HIV infections. Furthermore, we found that miR-19b can directly inhibit viral production in in-vitro HIV infected T cells. These results highlight the importance of miR-19b to control viral levels, which facilitate an understanding of human immunodeficiency virus pathogenesis and provide potential targets for improved immune intervention.

Deregulated MicroRNA-21 Expression in Monocytes from HIV-Infected Patients Contributes to Elevated IP-10 Secretion in HIV Infection.

Persistent activation and inflammation impair immune response and trigger disease progression in HIV infection. Emerging evidence supports the supposition that excessive production of interferon-inducible protein 10 (IP-10), a critical inflammatory cytokine, leads to immune dysfunction and disease progression in HIV infection. In this study, we sought to elucidate the cause of the upregulated production of IP-10 in HIV infection and explore the underlying mechanisms. Bolstering miR-21 levels using mimics resulted in the obvious suppression of lipopolysaccharide (LPS)-induced IP-10 in monocyte leukemia cells THP-1 and vice versa. The analysis of the primary monocytes of HIV patients revealed significantly less miR-21 than in healthy controls; this was opposite to the tendency of IP-10 levels in plasma. The secretion of IP-10 due to LPS stimulation was not affected by miR-21 modulation in the differentiated THP-1 macrophages (THP-1-MA). We found a novel switch, IFN-stimulated gene 15 (ISG15), which triggers the expression of IP-10 and is significantly upregulated during the differentiation of THP-1 into THP-1-MA. The inhibition of ISG15 can restore the regulation of IP-10 by miR-21. In summary, IP-10 expression in monocytes is regulated by miR-21, whereas in macrophages, this fine-tuning is attenuated by the enhanced expression of ISG15. This study paves the way to a comprehensive understanding of the molecular regulatory mechanism of IP-10, a key point in immune intervention strategy.

Transcriptomic meta-analysis identifies gene expression characteristics in various samples of HIV-infected patients with nonprogressive disease.

BACKGROUND: A small proportion of HIV-infected patients remain clinically and/or immunologically stable for years, including elite controllers (ECs) who have undetectable viremia (<50 copies/ml) and long-term nonprogressors (LTNPs) who maintain normal CD4+ T cell counts for prolonged periods (>10 years). However, the mechanism of nonprogression needs to be further resolved. In this study, a transcriptome meta-analysis was performed on nonprogressor and progressor microarray data to identify differential transcriptome pathways and potential biomarkers. METHODS: Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed genes (DEGs) in nonprogressors and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DEGs identified in the meta-analysis. Five microarray datasets (81 cases and 98 controls in total), including whole blood, CD4+ and CD8+ T cells, were collected for meta-analysis. RESULTS: We determined that nonprogressors have reduced expression of important interferon-stimulated genes (ISGs), CD38, lymphocyte activation gene 3 (LAG-3) in whole blood, CD4+ and CD8+ T cells. Gene ontology (GO) analysis showed a significant enrichment in DEGs that function in the type I interferon signaling pathway. Upregulated pathways, including the PI3K-Akt signaling pathway in whole blood, cytokine-cytokine receptor interaction in CD4+ T cells and the MAPK signaling pathway in CD8+ T cells, were identified in nonprogressors compared with progressors. In each metabolic functional category, the number of downregulated DEGs was more than the upregulated DEGs, and almost all genes were downregulated DEGs in the oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle in the three types of samples. CONCLUSIONS: Our transcriptomic meta-analysis provides a comprehensive evaluation of the gene expression profiles in major blood types of nonprogressors, providing new insights in the understanding of HIV pathogenesis and developing strategies to delay HIV disease progression.

Myeloid-Derived Suppressor Cells Associated With Disease Progression in Primary HIV Infection: PD-L1 Blockade Attenuates Inhibition.

OBJECTIVE: Events occurring during the initial phase of human immunodeficiency virus (HIV) infection are intriguing because of their dramatic impact on the subsequent course of the disease. In particular, the relationship between myeloid-derived suppressor cells (MDSCs) and HIV pathogenesis in primary infection remains unknown and the mechanism of MDSCs in HIV infection are incompletely defined. METHODS: The frequency of MDSC expression in patients with primary HIV infection (PHI) and chronic HIV infection was measured, and the association with disease progression was studied. Programmed death-ligand 1 (PD-L1) and galectin-9 (Gal-9) expression on MDSCs was measured and in vitro blocking experiments were performed to study the role of PD-L1 in MDSCs' inhibition. RESULTS: We found increased levels of HLA-DRCD14CD33CD11b granulocytic(G)-MDSCs in PHI individuals compared with normal controls, which correlated with viral loads and was negatively related to CD4 T-cell levels. When cocultured with purified G-MDSCs, both proliferation and interferon-γ secretion by T cell receptor (TCR)-stimulated CD8 T cells from HIV-infected patients were significantly inhibited. We also demonstrated that PD-L1, but not Gal-9, expression on HLA-DRCD14CD33CD11b cells increased during HIV infection. The suppressive activity of G-MDSCs from HIV-infected patients was attenuated by PD-L1 blockade. CONCLUSIONS: We found a significant increase in G-MDSCs in PHI patients that was related to disease progression and PD-L1 was used by MDSCs to inhibit CD8 T cells in HIV infection. Our data improve the understanding of HIV pathogenesis in PHI.