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IntroductionIn people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function.MethodsWe used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles.ResultsSoluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART.DiscussionPlasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

Background. Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Methods. Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. Results. CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P

Chronic activation of T cells is a hallmark of HIV-1 infection and plays an important role in disease progression. We previously showed that the engagement of the inhibitory receptor programmed death (PD)-1 on HIV-1-specific CD4(+) and CD8(+) T cells leads to their functional exhaustion in vitro. However, little is known about the impact of PD-1 expression on the turnover and maturation status of T cells during the course of the disease. In this study, we show that PD-1 is upregulated on all T cell subsets, including naive, central memory, and transitional memory T cells in HIV-1-infected subjects. PD-1 is expressed at similar levels on most CD4(+) T cells during the acute and the chronic phase of disease and identifies cells that have recently entered the cell cycle. In contrast, PD-1 expression is dramatically increased in CD8(+) T cells during the transition from acute to chronic infection, and this is associated with reduced levels of cell proliferation. The failure to downregulate expression of PD-1 in most T cells during chronic HIV-1 infection is associated with persistent alterations in the distribution of T cell subsets and is associated with impaired responses to IL-7. Our findings identify PD-1 as a marker for aberrant distribution of T cell subsets in HIV-1 infection.

The assembly of hepatitis C virus (HCV) is not well understood. We investigated HCV nucleocapsid assembly in vitro and the role of electrostatic/hydrophobic interactions in this process. We developed a simple and rapid in vitro assay in which the progress of assembly is monitored by measuring an increase in turbidity, thereby allowing the kinetics of assembly to be determined. Assembly is performed using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from Escherichia coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive charge of C1-82 rather than any specific basic residue that is important for the assembly process. Our in vitro assembly assay provides an easy and efficient means of screening for assembly inhibitors, and we have identified several inhibitory peptides that could represent a starting point for drug design.

Additional file 6. Poly-functional profiles of CM CCR6+ subsets upon long-term culture under Th17 versus Th1 polarizing conditions in vitro. FACS-sorted CM subsets were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro (Fig. 5). Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), four (yellow), and five (red) or no (purple) cytokines produced by T-cell subsets upon culture in vitro (mean ± SEM), together with pie charts summarizing the polyfunctional profiles (median relative contribution, n = 3).

Additional file 4. Superior Th17-lineage commitment of CM versus EM Th17 and Th1Th17-subsets. FACS-sorted Th17, Th1Th17 and Th1 subsets with CM (CD45RA−CCR7+) and EM (CD45RA−CCR7−) phenotype were cultured for 14 days under Th17-polarizing conditions, as in Fig. 5. Shown are the statistical analyses of the intracellular expression of IL-17A, IL-17F, IL-22, IFN-γ and TNF-α by distinct Th17-polarized CM (black bars) versus EM (white bars) Th17, Th1Th17 and Th1 subsets. Results (mean ± SEM) were generated with matched samples from n = 3 different subjects. Paired t-test p-values are indicated in the figures (CM versus EM).

Additional file 9. The four CCR6+ T-cell subsets isolated from HIV-infected subjects receiving ART preserve their Th17-polarizing profiles upon long term culture in vitro. (A) FACS-sorted Th17, Th1Th17 and Th1 subsets were stimulated with CD3/CD28 for four days then cultured in the presence of IL-2 (5 ng/ml) for an additional 9 days. At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs. (B) Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and IFNγ (n = 3). (C) Shown is the frequency of cytokine-expressing T-cells cultured long-term in vitro. Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p-values are indicated on the figures.

Additional file 7. Phenotypic stability versus plasticity of the four central memory CCR6+ T-cell subsets under Th17- or Th1-polarizing conditions. FACS-sorted CM subsets were cultured under Th17- and Th1-polarizing conditions for 14 days, as described in Fig. 5A, stained with CCR6, CCR4, and CXCR3Abs and analyzed by FACS. Shown are the statistical analyses for the expression of (A) CCR6 (B) CCR4, and (C) CXCR3, and the co-expression of (D) CCR6 and CCR4 (R6+R4+ phenotype) and (E) CCR6 and CXCR3 (R6+X3+ phenotype) on each subsets at day 0 (white bars) versus day 14 in culture under Th17- (black bars) and Th1-polarizing (grey bars) conditions. (A-E) The positivity gates where defined based on fluorescence minus one (FMO) controls. Results (mean ± SEM) were generated with matched subsets from n = 3 distinct subjects. Paired t-Test p-values are indicated on the graphs.

Additional file 5. Flow cytometry analysis of cytokine co-expression at single-cell level by CM subsets upon long-term culture under Th17 versus Th1 polarizing conditions. Central memory (CM) Th17, Th1Th17, CCR6+DN, CCR6+DP, and Th1 subsets were sorted and cultured for 14 days under Th17 or Th1 polarizing conditions, as described in Fig. 5. Shown are flow cytometry dot plots illustrating the co-expression of IL-17A with IL-17F, IL-22, IFN-γ or TNF-α for each Th17- or Th1-polarized subset. Results are from one donor representative of results generated with cells from three different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 1. Purity of flow cytometry-sorted memory CD4+ T-cell subsets. Total CD4+ T-cells were isolated from PBMCs of healthy individuals by negative selection using magnetic beads (Miltenyi). Cells were stained with a cocktail of fluorochrome-conjugated Abs and analyzed by polychromatic flow cytometry (see Supplemental Experimental Procedure). Memory (CD45RA−) cells lacking the lineage-specific markers CD8 (CD8+ T-cells), CD19 (B cells), and CD56 (NK cells) and with differential expression of CCR6, CCR4, and CXCR3 were sorted by flow cytometry (BDAria II) as follows: CCR6+CCR4+CXCR3− (Th17), CCR6+CCR4−CXCR3− (CCR6+DN), CCR6+CCR4+CXCR3+ (CCR6+DP), CCR6+CCR4−CXCR3+ (Th1Th17) and CCR6−CCR4−CXCR3+ (Th1). A viability staining was used to exclude dead cells. The positivity gates where defined based on fluorescence minus one (FMO) controls. Shown is (A) the gating strategy for the identification of different subsets on CD4+ T-cells sorted by MACS and (B) purity upon FACS sorting of different memory T-cell subsets. The percentage of each subset is indicated on the figures. Results are from one donor representative of experiments performed with cells from >10 different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 10. All four CCR6+ T-cell subsets carry integrated HIV-DNA in viremic untreated HIV-infected subjects. The four memory CCR6+ subsets as well as naive cells from PBMCs of (A) HIV+ recently infected untreated (RI) (n = 3) and were sorted by FACS. Levels of integrated HIV-DNA were quantified by nested real-time PCR (mean ± SD of triplicate wells).

Additional file 11. Poly-functional profiles of HIV-p24+ CCR6+ T-cell subsets of HIV-infected individuals receiving ART upon viral reactivation in vitro. HIV reservoir reactivation was performed as described in Suppl. Figure 6 legend. Briefly, at day 13, cells were stimulated with PMA/Ionomycin in the presence of Brefeldin A and intracellular staining was performed with cytokine (IFN-γ, IL-17A, IL-22, TNF-α) and HIV-p24 Abs. Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), and four (yellow), or no (purple) cytokines produced by HIV-p24+ Th17, Th1Th17, CCR6+DN, CCR6+DP and Th1 subsets (n = 3 CI on ART subjects). In contrast to CI on ART 5 and 6, for donor CI on ART 07 HIV reactivation was observed only for CCR6+DN (red).

Additional file 10. All four CCR6+ T-cell subsets carry integrated HIV-DNA in viremic untreated HIV-infected subjects. The four memory CCR6+ subsets as well as naive cells from PBMCs of (A) HIV+ recently infected untreated (RI) (n = 3) and were sorted by FACS. Levels of integrated HIV-DNA were quantified by nested real-time PCR (mean ± SD of triplicate wells).

Additional file 6. Poly-functional profiles of CM CCR6+ subsets upon long-term culture under Th17 versus Th1 polarizing conditions in vitro. FACS-sorted CM subsets were analyzed for the expression of lineage-specific cytokines upon Th17/Th1-polarization in vitro (Fig. 5). Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), four (yellow), and five (red) or no (purple) cytokines produced by T-cell subsets upon culture in vitro (mean ± SEM), together with pie charts summarizing the polyfunctional profiles (median relative contribution, n = 3).

Additional file 1. Purity of flow cytometry-sorted memory CD4+ T-cell subsets. Total CD4+ T-cells were isolated from PBMCs of healthy individuals by negative selection using magnetic beads (Miltenyi). Cells were stained with a cocktail of fluorochrome-conjugated Abs and analyzed by polychromatic flow cytometry (see Supplemental Experimental Procedure). Memory (CD45RA−) cells lacking the lineage-specific markers CD8 (CD8+ T-cells), CD19 (B cells), and CD56 (NK cells) and with differential expression of CCR6, CCR4, and CXCR3 were sorted by flow cytometry (BDAria II) as follows: CCR6+CCR4+CXCR3− (Th17), CCR6+CCR4−CXCR3− (CCR6+DN), CCR6+CCR4+CXCR3+ (CCR6+DP), CCR6+CCR4−CXCR3+ (Th1Th17) and CCR6−CCR4−CXCR3+ (Th1). A viability staining was used to exclude dead cells. The positivity gates where defined based on fluorescence minus one (FMO) controls. Shown is (A) the gating strategy for the identification of different subsets on CD4+ T-cells sorted by MACS and (B) purity upon FACS sorting of different memory T-cell subsets. The percentage of each subset is indicated on the figures. Results are from one donor representative of experiments performed with cells from >10 different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 11. Poly-functional profiles of HIV-p24+ CCR6+ T-cell subsets of HIV-infected individuals receiving ART upon viral reactivation in vitro. HIV reservoir reactivation was performed as described in Suppl. Figure 6 legend. Briefly, at day 13, cells were stimulated with PMA/Ionomycin in the presence of Brefeldin A and intracellular staining was performed with cytokine (IFN-γ, IL-17A, IL-22, TNF-α) and HIV-p24 Abs. Shown are bar graph representations generated with SPICE software for all possible combinations of one (blue), two (green), three (orange), and four (yellow), or no (purple) cytokines produced by HIV-p24+ Th17, Th1Th17, CCR6+DN, CCR6+DP and Th1 subsets (n = 3 CI on ART subjects). In contrast to CI on ART 5 and 6, for donor CI on ART 07 HIV reactivation was observed only for CCR6+DN (red).

Additional file 5. Flow cytometry analysis of cytokine co-expression at single-cell level by CM subsets upon long-term culture under Th17 versus Th1 polarizing conditions. Central memory (CM) Th17, Th1Th17, CCR6+DN, CCR6+DP, and Th1 subsets were sorted and cultured for 14 days under Th17 or Th1 polarizing conditions, as described in Fig. 5. Shown are flow cytometry dot plots illustrating the co-expression of IL-17A with IL-17F, IL-22, IFN-γ or TNF-α for each Th17- or Th1-polarized subset. Results are from one donor representative of results generated with cells from three different donors. The positivity gates where defined based on fluorescence minus one (FMO) controls.

Additional file 4. Superior Th17-lineage commitment of CM versus EM Th17 and Th1Th17-subsets. FACS-sorted Th17, Th1Th17 and Th1 subsets with CM (CD45RA−CCR7+) and EM (CD45RA−CCR7−) phenotype were cultured for 14 days under Th17-polarizing conditions, as in Fig. 5. Shown are the statistical analyses of the intracellular expression of IL-17A, IL-17F, IL-22, IFN-γ and TNF-α by distinct Th17-polarized CM (black bars) versus EM (white bars) Th17, Th1Th17 and Th1 subsets. Results (mean ± SEM) were generated with matched samples from n = 3 different subjects. Paired t-test p-values are indicated in the figures (CM versus EM).

Additional file 9. The four CCR6+ T-cell subsets isolated from HIV-infected subjects receiving ART preserve their Th17-polarizing profiles upon long term culture in vitro. (A) FACS-sorted Th17, Th1Th17 and Th1 subsets were stimulated with CD3/CD28 for four days then cultured in the presence of IL-2 (5 ng/ml) for an additional 9 days. At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs. (B) Shown are flow cytometry dot plots illustrating the co-expression of IL-17A and IFNγ (n = 3). (C) Shown is the frequency of cytokine-expressing T-cells cultured long-term in vitro. Results (mean ± SEM) were generated with matches samples from n = 3 different donors. Paired t-Test p-values are indicated on the figures.

Additional file 7. Phenotypic stability versus plasticity of the four central memory CCR6+ T-cell subsets under Th17- or Th1-polarizing conditions. FACS-sorted CM subsets were cultured under Th17- and Th1-polarizing conditions for 14 days, as described in Fig. 5A, stained with CCR6, CCR4, and CXCR3Abs and analyzed by FACS. Shown are the statistical analyses for the expression of (A) CCR6 (B) CCR4, and (C) CXCR3, and the co-expression of (D) CCR6 and CCR4 (R6+R4+ phenotype) and (E) CCR6 and CXCR3 (R6+X3+ phenotype) on each subsets at day 0 (white bars) versus day 14 in culture under Th17- (black bars) and Th1-polarizing (grey bars) conditions. (A-E) The positivity gates where defined based on fluorescence minus one (FMO) controls. Results (mean ± SEM) were generated with matched subsets from n = 3 distinct subjects. Paired t-Test p-values are indicated on the graphs.