Demographic and Clinical Characteristics of Patients and Control groups.

a<p>HIV uninfected samples are blood from commercial source</p>b<p>Data are for patients who received antiretrovirals, patients with natural virus suppression (NVS) and chronic low viremia (VIR) and are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088884#pone.0088884-Hardy1" target="_blank">[50]</a>.</p

Exhausted CD56+ CD8 T lymphocytes in treated HIV infection.

<p>The expression of Tim-3 on T cells identified exhausted T lymphocytes. (A) A higher proportion of CD56+ CD8 T cells express Tim-3 compared to the CD56- subset. (B) Cumulative data from all individuals irrespective of disease status shows significantly higher Tim-3 expression by CD56+ CD8 T cells (C) The proportion of CD56+CD8+ T cells that are exhausted as judged by their Tim-3 expression were significantly higher in HIV+ART but not in elite controllers when compared to uninfected group. (D) The levels of Tim-3 on CD56- CD8 T cells is comparable in all HIV- and HIV+ groups.</p

Elite controllers have higher levels of perforin/GranzymeB+CD56+ CD8 T lymphocytes.

<p>Intracellular expression of preformed perforin (A) and Granzyme B (B) is significantly higher on gated CD8 T lymphocytes from elite controllers and low viremia groups compared to uninfected controls or HIV+ART subjects. The level of CD56 expression on perforin (C) or Granzyme B (D) expressing CD8 T lymphocytes was significantly reduced in HIV+ ART treated and low viremia groups but not in elite controllers. Results in panels E-G show perforin upregulation in response to stimulation with HIV gag peptides measured with DG48 clone of anti-perforin antibody. (E) Both elite controller and low viremia group have significantly higher production of perforin upregulation in response to stimulation compared with ART treated individuals. (F). Representative sample from each infected group showing majority of CD56+ CD8 T cells upregulated perforin in response to stimulation while a minority of CD56- CD8 T cells upregulate perforin. Cells are gated on CD8 T lymphocytes. (G) Combined results from all infected groups show proportion of CD56 fraction of CD8 T cells upregulating perforin in response to stimulation with HIV peptides is significantly higher than proportion of CD56- CD8 T cell that upregulate perforin. Significantly greater proportions of CD56+ CD8 T cells degranulate (H) as well as produce IFNγ (I) in response to stimulation with HIV gag peptides. Results in G, H and I show show comparison of CD56+ and CD56- subsets irrespective of the patient's disease status.</p

15d-PGJ2, but not rosiglitazone, suppressed cytokine production, degranulation and cytotoxicity functions of Vδ2 T cell.

<p>(A) Freshly isolated PBMC contained 1–10% of Vδ2 T cells (left panel); after 10 to 14 days of culture with IPP plus IL-2, the percentage of Vδ2 T cells was more than 90% (right panel). (B, C and D) Vδ2 T cells were treated with 15d-PGJ2 or rosiglitazone at various concentrations for 1 hour and then washed and stimulated with IPP (50 µM). After stimulating for 4 hours, the levels of IFN-γ (B) or TNF-α (C) in cell-free supernatant were detected by antigen capture ELISA. The experiments were done in triplicate and statistical tests compared drug and vehicle (DMSO). CD107a expression (D) was analyzed by flow cytometry. (E and F) Vδ2 T cells were pretreated with 15d-PGJ2 at various concentrations for 1 hour. The cytotoxicity of Vδ2 T cell against Daudi (E) or TU167 (F) was evaluated at different E∶T ratios in triplicate. The statistical significance of specific lysis compared with a drug vehicle (DMSO) control at E∶T = 5∶1 was analyzed. (G and H) Vδ2 T cells were pretreated with rosiglitazone at various concentrations for 1 hour. The cytotoxicity of Vδ2 T cell against Daudi (G) or TU167 (H) was evaluated at different E∶T ratios in triplicate. (I) PBMC was stimulated with PMA (10 ng/ml) and ionomycin (1 µM) for 4 h. IL-17 production in different cell type was detected by flow cytometry. *, P<0.05; **, P<0.01; ***, P<0.001. Data are representative of three independent experiments using different donors.</p

Transcription factors and MAPK signaling in CD56 subset.

<p>EOMES and T-bet control cytotoxic function of effector T cells. (A) Majority of CD56+ CD8 T cells expressed T-bet and/or EOMES and were double positive for these transcription factors, whereas a significant proportion of CD56- CD8 T cells were double negative for EOMES and T-bet. The representative plots show the trends presend in CD56+ or CD56- subsets from all patient groups irrespective of disease status (B) NVS group had highest frequency of T-bet expressing CD8 T cells whereas there was no significant difference in EOMES positive CD8 T cells among different cohorts of HIV infected patients. (C) Overall, the CD56+ fraction of CD8 T lymphocytes had higher levels of Erk phosphoryation upon PMA+inomycin stimulation compared with the CD56- CD8 T lymphocytes when samples from all groups were combined for analysis. CD56+ or CD56- CD8 T lymphocytes showed similar levels of Erk phosphorylation in all infected and control groups.</p

15d-PGJ2 and rosiglitazone suppressed IL2-induced phophorylation of STAT5 in Vδ2 T cells.

<p>Purified Vδ2 T cells from fresh PBMC were pretreated with 15d-PGJ2 (10 µM) or rosiglitazone (50 µM) for 1 hour, then incubated with IL2 (100 U/ml) for 15 minutes. The phosphorylated STAT5 was stained with a specific antibody permeabilized cells and detected by flow cytometry.</p

Preserved CD56+ CD8 T lymphocytes in elite controllers.

<p>(A, B) Higher expression of CD56 on CD8dim T compared with CD8bright T lymphocytes. (C) Representative samples shows CD56+ CD8 T lymphocytes are mostly effector memory type whereas (D) CD56- CD8 T lymphocytes have a significant subset of naïve and central memory and type cells. (E) Both CD56+ and CD56- subset of CD8 T cells have similar frequency of HIV tetramer binding cells. Cells from an HLAB5701 HIV infected individual were stained with TW10 tetramer. Cells are gated on CD8+ CD3+ lymphocytes. (F) Significantly elevated CD8 T cell frequencies in both ART treated and low viremia (VIR) groups but not in elite controllers (NVS). (G) The frequency of CD56+ CD8+ T lymphocytes is significantly reduced in HIV+ ART treated group compared with uninfected controls. NVS (elite controllers) did not show this defect and have normal levels of CD56 expression on CD8 T cells. (H) Analysis of CD8bri (H) or CD8dim (I) populations of T cells show that NVS group has superior levels of CD56 subset among both populations whereas ART group has lowest levels of CD8bri cells expressing CD56. VIR group has high frequency of CD56 expressing CD8bri but not CD8dim cells when compared with ART patients. For F-I, Kruksal-Wallis test followed by Dunn's test for multiple correction was used to analyze the four groups.</p

Primary and expanded Vδ2 T cells express PPARγ.

<p>The expression of PPARγ in both primary and expanded Vδ2 T cells was examined by flow cytometry using intracellular staining (A) or western blotting (B). Data are representative of two independent experiments.</p

15d-PGJ2 suppressed Vδ2 T cell functions by inhibiting Erk activation.

<p>The expanded Vδ2 T cells were rested, incubated in fresh medium for 24 hours without stimulation. (A) The cells were treated with drug vehicle (DMSO), 15d-PGJ2 (10 µM) or rosiglitazone (50 µM) for 1 hour, then stimulated with or without IPP (15 µM). After 30 minutes, cells were collected for western blotting analyses. (B) The cells were treated with drug vehicle (DMSO) or U0126 for 1 hour, then stimulated with or without IPP (15 µM). After 30 minutes, cells were collected for western blotting analyses. (C and D) Vδ2 T cells were treated with drug vehicle (DMSO) or U0126 for 1 hour, then washed and stimulated with IPP. After 4 hours, the levels of cell-free IFN-γ (C) or TNF-α (D) were detected by antigen capture ELISA. The experiments were done in triplicate and statistical tests compared drug with vehicle (DMSO). (E and F) Vδ2 T cells were pretreated with U0126 for 1 hour. The cytotoxicity of Vδ2 T cells against Daudi (E) or TU167 (F) was evaluated at different E∶T ratios. Statistical tests of specific lysis compared drug with vehicle (DMSO) control at E∶T = 5∶1 was analyzed. (G) Fresh isolated PBMC was treated with U0126 at various concentrations for 1 hour, then stimulated with IPP (15 µM) plus IL2 (100 U/ml). Cells were cultured for 10 days by adding IL2 every 3 days. Vδ2 T cell frequencies were detected every 3 days. The experiments were done in triplicate. Statistical tests compared drug with vehicle (DMSO). *, P<0.05; **, P<0.01; ***, P<0.001. Data are representative of three independent experiments with different donors.</p

IL-15 restores CD56 on CD8 T cells from HIV infected patients.

<p>(A) Most CD8 T cells and almost all CD56 + CD8 T cells express the common gamma chain (γ_c) (or CD132). Representative plots (from left ND, PD, NVS, VIR) show there was no significant downregulation of this receptor on CD8 T lymphocytes from any infected group (B) Culture of PMBC in IL-15 resulted in a significant upregulation of CD56 on CD8 T lymphocytes at day 12 relative to start of culture in infected and uninfected samples.</p