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Supplementary methods
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Multivariate analysis
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Multivariate analysis was used to compare the predictive strength of CD4+Glut1+ percentage with
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established predictors of HIV disease progression. CD4 count, total CD4 percentage and viral load
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were selected as the respective outcome variables and the different candidate immune activation
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markers were included as the predictor variables. Significant markers in the univariable analyses
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were entered in the multivariable model and the final model was derived through a process of
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stepwise elimination.
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Overexpression and detection of Glut1 on HEK293T cells
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HEK293T cells were grown in RPMI 1640 medium, supplemented with 10% fetal calf serum (Gibco®),
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2mM L-glutamine (Gibco®), 100 U/ml penicillin (Gibco®), and 100 μg/ml streptomycin (Gibco®). A
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total of 5 x 104 cells were seeded per well in a 96-well plate and cultured for 24h. Cells were then
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transfected using Lipofectamine 2000 (Invitrogen) with 0.25 µg of either pQCXIP-huGlut1 plasmid (A
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kind gift from professor Steven M. Anderson, The Rockefeller University, USA) or empty vector
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pQCXIP. At 48 h post-transfection, cells were harvested, washed twice in 1×PBS and stained with
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FITC-labelled anti-Glut-1 antibody (R&D) for 30 min at 4°C. Cells were subsequently washed two
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times with 1×PBS and analyzed by flow cytometry (BD FACSCanto™ Flow Cytometer).
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Overexpression, Intracellular and cell surface detection of Glut1 in NIH3T3
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The coding sequence of human Glut1 was restriction digested and purified from R&D System human
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Glut1 cDNA (Cat #RDC0128) construct and sub cloned into our in-house retroviral construct pR-IRES-
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eGFP [1]. NIH3T3 cells were transfected with 1.5 µg of either Glut1-pR-IRES-eGFP or vector-only
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control using Life Technology's Lipofectamine 2000 (Cat #11668) as per manufacturer's
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recommendation. 48 hours after transfection, cells were trypsinised and single cell suspensions were
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made. Cells are permeabilised with BD's Cytofix/Cytoperm (Cat #554714) per manufacturer's
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recommendations then stained with R&D system's anti-human Glut1 antibody (Cat #MAB1418,
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clone 202915) at the recommended concentration of 2.5 µg per million cells. The primary antibody
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was detected using BD goat polyclonal anti-mouse-IgG multiple absorption APC (Cat #550826). Data
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was acquired on a BD LSRII flow cytometer.
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Western blot analysis of Glut1
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Protein was extracted from HEK293T cells transfected with either pQCXIP-huGlut1 or empty pQCXIP
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vector. A total of 1 million cells were lysed in RIPA buffer supplemented with protease inhibitors.
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Protein concentration in the cell lysates was determined by Bradford assay and a total of 30 µg of
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proteins were run in a SDS-PAGE in Laemmli loading buffer as previously described [2]. Glut1 was
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detected using Glut1c-term antibody (Abcam).
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Supplementary figure legends
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Figure S1. Glut1 mRNA expression is higher in CD4+ T cells from HIV+/naïve subjects. (A) Glut1
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mRNA expression in HIV-, HIV+ and HIV+/cART subjects. (B) Spearman’s correlation between Glut1
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mRNA expression in CD4+ T cells and the percentage of CD4+Glut1+ T cells. Red and blue dots
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represent HIV+/naïve and HIV+/cART subjects, respectively. (C) Spearman’s correlation between the
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number of years on cART and the percentage of CD4+Glut1+ T cells. The non-parametric Mann-
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Whitney T test was used to evaluate significant differences between the median values of each
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group. Horizontal bars represent median values.
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Figure S2. The relationship between the percentage of CD4+ T cell subpopulations and the
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percentage of CD4+Glut1+ T cells. (A) Percentage of CD4+ subpopulations in HIV- and HIV+/naïve
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subjects. (B) The percentage of CD4 memory (M) T cells is inversely correlated with percentage of
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CD4+Glut1+ cells. (C) The percentage of CD4 effector (E) and effector memory (EM) T cells is
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correlated positively with the percentage of CD4+Glut1+ T cells. Spearman’s correlations were
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conducted across the two subject groups. (D) Absolute CD4+Glut1+ T cell count in HIV+/naïve and
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HIV+/cART subjects. The non-parametric Mann-Whitney T test was used to evaluate significant
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differences between the median values of each group. Horizontal bars represent median values.
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Figure S3. Reactivity of Glut1 antibodies on cells transfected with Glut1-containing lentiviral
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vectors. (A) HEK293T cells transfected with pQCXIP–huGlut1 vector and stained with IgG2b-FITC
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isotype (black line) or Glut1-FITC antibody (grey line). (B) HEK293T cells transfected with empty
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(pQCXIP) vector and stained with IgG2b-FITC isotype (black line) or Glut1-FITC antibody (grey line).
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(C) Western blot analysis of Glut1 using protein extracted from HEK293T cells transfected with
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pQCXIP-huGlut1 (lane 1) or with empty (pQCXIP) vector (lane 2). Glut1 was detected using a Glut1c-
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antibody (Abcam). (D) Intracellular detection of Glut1 on NIH3T3 cells transfected with a
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lentiviral vector expressing Glut1. Dual analysis of Glut1 and eGFP. (E) Histogram illustrating
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fluorescent shift using R&D System Glut1-APC conjugated antibody in permeabilized cells
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transfected with empty (vector only control) or huGlut1-expressing lentiviral vector.
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Figure S4. Cell surface and intracellular expression of Glut1 on cell line. (A) SSC and FSC plot of
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Jurkat cells. (B) Intracellular (left) and cell surface (right) expression on Glut1 on paraformaldehyde-
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fixed Jurkat cells. (C) Co-expression of cell surface and c-terminal Glut1 in Jurkat cells. (D) SSC and
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FSC plot of N2a cells. (E) Intracellular (left) and cell surface (right) expression on Glut1 in N2a cells.
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(F) Co-expression of cell surface and c-terminal Glut1 in N2a cells.
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Figure S5. Kinetics of expression of different activation markers on CD4+Glut1+ T cells. (A) The
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percentage of CD4+Glut1+ T cells was assessed at the indicated times following activation of PBMCs
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with anti-CD3/CD28 microbeads (1:1 cell: bead ratio). The data represent mean (SD) of results from
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four HIV- subjects. (B) The percentage of CD4+Glut1+ T cells that expressed different activation
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markers was assessed over time on activated PBMCs. The data represent mean (SD) of results from
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four HIV- subjects. (C) Flow cytometric dot plot showing the co-expression of Glut1 with markers of T
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cell activation on CD4+ T cells.
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Figure S6. Relationship between percentage CD3+CD4+ T cells, CD4 T cell count and markers of
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immune activation in HIV-infected subjects. (A) Comparative relationship between the percentage of
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CD4+Glut1+ T cells and markers of CD8+ T cell activation, and the percentage of CD3+CD4+ T cells in
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HIV+/naïve, and (B) HIV+/cART subjects. (C) Comparative relationship between the percentage of
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CD4+Glut1+ T cells and markers of CD8+ T cell activation, and HIV viral load in HIV+/naïve subjects
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