To obtain a comprehensive scenario of T cell profiles and synergistic immune responses, we performed single-cell RNA sequencing (scRNA-seq) on the peripheral T cells of 14 individuals with chronic ...human immunodeficiency virus 1 (HIV-1) infection, including nine treatment-naive (TP) and eight antiretroviral therapy (ART) participants (of whom three were paired with TP cases), and compared the results with four healthy donors (HD). Through analyzing the transcriptional profiles of CD4
and CD8
T cells, coupled with assembled T cell receptor sequences, we observed the significant loss of naive T cells, prolonged inflammation, and increased response to interferon-α in TP individuals, which could be partially restored by ART. Interestingly, we revealed that CD4
and CD8
Effector-GNLY clusters were expanded in TP cases, and persistently increased in ART individuals where they were typically correlated with poor immune restoration. This transcriptional dataset enables a deeper understanding of the pathogenesis of HIV-1 infection and is also a rich resource for developing novel immune targeted therapeutic strategies.
Background
Targeting immune checkpoints for HIV treatment potentially provides a double benefit resulting from the ability to restore viral-specific CD8
+
T-cell functions and enhance HIV production ...from reservoir cells. Despite promising pre-clinical data, PD-1 blockade alone in HIV-1-infected patients with advanced cancer has shown limited benefits in controlling HIV, suggesting the need for additional targets beyond PD-1. CD39 and PD-1 are highly co-expressed on CD8
+
T cells in HIV-1 infection. However, the characteristics of CD39 and PD-1 dual-positive CD8
+
T-cell subsets in chronic HIV-1 infection remain poorly understood.
Methods
This study enrolled 72 HIV-1-infected patients, including 40 treatment naïve and 32 ART patients. A total of 11 healthy individuals were included as controls. Different subsets of CD8
+
T cells defined by CD39 and/or PD-1 expression were studied by flow cytometry. The relationships between the frequencies of the different subsets and parameters indicating HIV-1 disease progression were analyzed. Functional (i.e., cytokine secretion, viral inhibition) assays were performed to evaluate the impact of the blockade of adenosine and/or PD-1 signaling on CD8
+
T cells.
Results
The proportions of PD-1
+
, CD39
+
, and PD-1
+
CD39
+
CD8
+
T cells were significantly increased in treatment naïve patients but were partially lowered in patients on antiretroviral therapy. In treatment naïve patients, the proportions of PD-1
+
CD39
+
CD8
+
T cells were negatively correlated with CD4
+
T-cell counts and the CD4/CD8 ratio, and were positively correlated with viral load. CD39
+
CD8
+
T cells expressed high levels of the A2A adenosine receptor and were more sensitive to 2-chloroadenosine-mediated functional inhibition than their CD39
-
counterparts.
In vitro
, a combination of blocking CD39/adenosine and PD-1 signaling showed a synergic effect in restoring CD8
+
T-cell function, as evidenced by enhanced abilities to secrete functional cytokines and to kill autologous reservoir cells.
Conclusion
In patients with chronic HIV-1 infection there are increased frequencies of PD-1
+
, CD39
+
, and PD-1
+
CD39
+
CD8
+
T cells. In treatment naïve patients, the frequencies of PD-1
+
CD39
+
CD8
+
T cells are negatively correlated with CD4
+
T-cell counts and the CD4/CD8 ratio and positively correlated with viral load. Combined blockade of CD39/adenosine and PD-1 signaling
in vitro
may exert a synergistic effect in restoring CD8
+
T-cell function in HIV-1-infected patients.
HIV replication can be inhibited by CXCR5+CD8 T cells (follicular cytotoxic T cell TFC) which transfer into B‐cell follicles where latent HIV infection persists. However, how cytokines affect TFC ...remain unclear. Understanding which cytokines show the ability to affect TFC could be a key strategy toward curing HIV. Similar mechanisms could be used for the growth and transfer of TFCs and follicular helper T (TFH) cells; as a result, we hypothesized that cytokines IL‐6, IL‐21, and transforming growth factor‐β (TGF‐β), which are necessary for the differentiation of TFH cells, could also dictate the development of TFCs. In this work, lymph node mononuclear cells and peripheral blood mononuclear cells from HIV‐infected individuals were cocultured with IL‐6, IL‐21, and TGF‐β. We then carried out T‐cell receptor (TCR) repertoire analysis to compare the differences between CXCR5– and CXCR5+CD8 T cells. Our results showed that the percentage and function of TFC can be enhanced by stimulation with TGF‐β. Besides, TGF‐β stimulation enhanced the diversity of TCR and complementarity‐determining region 3 sequences. HIV DNA showed a negative correlation with TFC. The use of TGF‐β to promote the expression of CXCR5+CD8 T cells could become a new treatment approach for curing HIV.
Chronic inflammation and T cell dysregulation persist in individuals infected with human immunodeficiency virus type 1 (HIV-1), even after successful antiretroviral treatment. The mechanism involved ...is not fully understood. Here, we used Olink proteomics to comprehensively analyze the aberrant inflammation-related proteins (IRPs) in chronic HIV-1-infected individuals, including in 24 treatment-naïve individuals, 33 immunological responders, and 38 immunological non-responders. T cell dysfunction was evaluated as T cell exhaustion, activation, and differentiation using flow cytometry. We identified a cluster of IRPs (cluster 7), including CXCL11, CXCL9, TNF, CXCL10, and IL18, which was closely associated with T cell dysregulation during chronic HIV-1 infection. Interestingly, IRPs in cluster 5, including ST1A1, CASP8, SIRT2, AXIN1, STAMBP, CD40, and IL7, were negatively correlated with the HIV-1 reservoir size. We also identified a combination of CDCP1, CXCL11, CST5, SLAMF1, TRANCE, and CD5, which may be useful for distinguishing immunological responders and immunological non-responders. In conclusion, the distinct inflammatory milieu is closely associated with immune restoration of T cells, and our results provide insight into immune dysregulation during chronic HIV-1 infection.
(1) Background: The objective of this study was to investigate the prevalence of genetic diversity and drug resistance mutations among people living with HIV (PLWH) attending clinics in Beijing. (2) ...Methods: A retrospective analysis was conducted on PLWH admitted to the Fifth Medical Center of People's Liberation Army (PLA) General Hospital between 1 March 2013 and 31 July 2020. The participants were analyzed for pretreatment drug resistance (PDR) and acquired drug resistance (ADR). Nested polymerase chain reaction (PCR) was utilized to amplify the pol gene from plasma RNA samples obtained from the participants. Genotypic and HIV drug resistance were determined using the Stanford University HIV Drug Resistance Database. Univariate and multifactorial logistic analyses were used to assess the risk factors for PDR. (3) Results: The overall prevalence rates of PDR and ADR were 12.9% and 27.8%, respectively. Individuals treated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) exhibited the highest prevalence of mutations. Specific mutation sites, such as V179D for NNRTIs and M184V and K65R for nucleoside reverse transcriptase inhibitors (NRTIs), were identified as prevalent mutations. Individuals treated with efavirenz (EFV) and nevirapine (NVP) were found to be susceptible to developing resistance. The multifactorial regression analyses indicated that the factors of circulating recombination form (CRF) genotype CRF07-BC and a high viral load were associated with an increased risk of PDR. CRF01-AE and CRF07-BC were the most prevalent HIV genotypes in our study. (4) Conclusions: The distribution of HIV genotypes in Beijing is complex. There is a need for baseline screening for HIV drug resistance among ART-naive individuals, as well as timely testing for drug resistance among ART-experienced individuals.
Whether varying CD8 counts influence the human immunodeficiency virus (HIV) reservoir and CD4 restoration in patients with CD4 counts ≥ 500 cells/μL after long-term antiretroviral therapy (ART) ...remains unknown. In this study, we analyzed relationships between CD8 levels and viral reservoir decay or CD4 recovery in immune restored patients on long-term ART.
Chronic HIV-infected patients who received 5 years of ART with CD4 counts ≥ 500 cells/μL were grouped according to CD8 counts: CD8 <500 (Group 1), 500-1,000 (Group 2), and ≥1,000 cells/μL (Group 3). CD4 recovery, viral decay, CD8 T-cell function, and their correlations were analyzed during ART among the three groups.
Dynamics of viral decay and CD4 recovery were different among the three groups. Both viral decay and CD4 recovery were higher in Group 3 than the other two groups after 5 years of ART, mainly during years 3-5 of ART. Higher expression levels of Ki67 while PD-1 levels were lower on CD8 T-cells in Group 3 compared with the other groups, and Group 3 showed stronger CD8 T-cells functional capacity after 3 years of ART. Reduced HIV DNA levels and increased CD4 counts between years 3 and 5 of ART were positively correlated with CD8 counts and function.
High CD8 counts are beneficial for persistent viral decay and CD4 recovery in immune restored patients during long-term ART.
People living with human immunodeficiency virus (PLWH) are a vulnerable population with a higher risk of severe coronavirus disease 2019 (COVID-19); therefore, vaccination is recommended as a ...priority. Data on viral reservoirs and immunologic outcomes for PLWH breakthrough infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently limited. In this study, we investigated the effects of SARS-CoV-2 breakthrough infection on hematological parameters, human immunodeficiency virus (HIV) reservoir size, and T-cell recovery in PLWH receiving antiretroviral therapy (ART) after SARS-CoV-2 booster vaccination. The results indicated that during breakthrough infection, booster vaccination with homologous and heterologous vaccines was safe in PLWH after receiving two doses of inactivated vaccination. The absolute CD4 counts decreased in the heterologous group, whereas the CD8 counts decreased in the homologous booster group after breakthrough infection in PLWH. Breakthrough infection increased HIV reservoirs and was associated with increased T-cell activation in PLWH who received virally suppressed ART and a 3-dose vaccination. According to our data, the breakthrough infection of SARS-CoV-2 may put PLWH at a greater risk for increased HIV reservoirs, even if these individuals were virally suppressed with ART after 3-dose SARS-CoV-2 vaccination.
Mucosal-associated invariant T (MAIT) cells are systemically depleted in human immunodeficiency virus type 1 (HIV-1) infected patients and are not replenished even after successful combined ...antiretroviral therapy (cART). This study aimed to identify the mechanism underlying MAIT cell depletion.
In the present study, we applied flow cytometry, single-cell RNA sequencing and immunohistochemical staining to evaluate the characteristics of pyroptotic MAIT cells in a total of 127 HIV-1 infected individuals, including 69 treatment-naive patients, 28 complete responders, 15 immunological non-responders, and 15 elite controllers, at the Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.
Single-cell transcriptomic profiles revealed that circulating MAIT cells from HIV-1 infected subjects were highly activated, with upregulation of pyroptosis-related genes. Further analysis revealed that increased frequencies of pyroptotic MAIT cells correlated with markers of systemic T-cell activation, microbial translocation, and intestinal damage in cART-naive patients and poor CD4
T-cell recovery in long-term cART patients. Immunohistochemical staining revealed that MAIT cells in the gut mucosa of HIV-1 infected patients exhibited a strong active gasdermin-D (GSDMD, marker of pyroptosis) signal near the cavity side, suggesting that these MAIT cells underwent active pyroptosis in the colorectal mucosa. Increased levels of the proinflammatory cytokines interleukin-12 (IL-12) and IL-18 were observed in HIV-1 infected patients. In addition, activated MAIT cells exhibited an increased pyroptotic phenotype after being triggered by HIV-1 virions, T-cell receptor signals, IL-12 plus IL-18, and combinations of these factors, in vitro.
Activation-induced MAIT cell pyroptosis contributes to the loss of MAIT cells in HIV-1 infected patients, which could potentiate disease progression and poor immune reconstitution.
Neutrophils are characterized by their heterogeneity. They fight against pathogens and are involved in tissue injury repair and immune system regulation. Neutrophils have an extremely short life span ...in the peripheral blood and undergo aging after being released from the bone marrow. The over-aggregation of aged neutrophils is associated with phenotypical and functional changes. Here, we aimed to investigate the dynamics of neutrophil aging and its relationship with T cell exhaustion in HIV-1 infection, as they are not well understood. In this study, we enrolled 23 treatment naïve (TN) patients, 23 individuals that had received antiretroviral therapy (ART), and 21 healthy controls (HC). In these cohorts, we measured the degree of neutrophil aging, and its possible correlation with T cell dysfunction. In TN patients, peripheral neutrophils showed a more distinct aging phenotype and were over-activated compared to those in ART-treated patients. The degree of neutrophil aging was positively correlated with HIV-1 RNA viral load and negatively correlated with CD4+ T cell count. Moreover, aged neutrophils had impaired reactive oxygen species (ROS) production after lipopolysaccharide (LPS) stimulation, and were characterized by increased PD-L1 and arginase-1 expression in a time-dependent manner. Aged neutrophils demonstrated an increased inhibition of IFN-γ and TNF-α secretion by CD8+ T cell compared to non-aged neutrophils. The inhibition effect could be partially reversed by blocking PD-L1 and arginase-1
in vitro
, and LPS was identified as an important activator of neutrophil aging. These results provide evidence that dampening neutrophil aging may provide a novel approach to recover T cell dysfunction in patients with HIV-1 infection.
Treg cells represent important viral reservoirs during chronic HIV infection. CD39 is closely involved in Treg-mediated immunosuppressive effects. However, CD39 expression on nTregs and mTregs and a ...relationship with HIV DNA levels during HIV infection is still unclear. In this study, we analyzed the distribution of HIV DNA in Treg subsets and the association between HIV DNA and CD39 expression on Treg subsets.
Sixty-two HIV-infected patients with different HIV stages and 14 uninfected individuals were enrolled. nTregs (CD4
CD25
CD127
CD45RO
) and mTregs (CD4
CD25
CD127
CD45RO
) were isolated by magnetic selection and flow cytometric sorting. HIV DNA was quantified by real-time polymerase chain reaction (PCR). CD39 expression on nTregs and mTregs was analyzed by flow cytometry.
Higher levels of HIV DNA were detected in mTregs than those in nTregs during chronic HIV infection. The frequency of CD39
nTregs and HIV DNA levels in nTregs were increased in patients with advanced HIV infection. Furthermore, HIV DNA levels in nTregs correlated positively with CD39
nTreg frequency. CD39
nTreg frequency was also increased in immune non-responders.
mTregs and nTregs are both important reservoirs of virus during chronic HIV infection and HIV DNA levels increase in nTregs in patients with advanced HIV infection. We observed increased frequency of CD39
nTregs and HIV DNA levels in nTregs in patients with advanced HIV infection. HIV DNA levels in nTregs correlated positively with CD39
nTreg frequency.
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