Fig 2: Glut1 cell surface expression on CD4+ T cells is associated with markers of proliferation and activation. (A) Representative flow cytometric dot plots of PBMCs from an HIV+/cART subject. Lymphocytes (circled) were defined using side scatter (SSC) and forward scatter (FSC) characteristics. The gating strategy shows T cells defined based on CD4 and CD8 surface expression. A representative Glut1‐isotype, and Glut1 antibody staining on CD4+ T cells in peripheral blood from HIV+/cART subjects. (B) Percentage of CD4+Glut1+ T cells in peripheral blood from HIV‐negative, HIV+/naive, and HIV+/cART subjects (left panel). Same subjects as in left panel showing percentages of CD4+Glut1+ T cells before and during cART (right panel). (C) Representative histogram (left panel) and aggregate plot (right panel) of HLA‐DR expression on CD4+Glut1+ and CD4+Glut1− T cell from HIV+ subjects. (D) Representative histogram (left panel) and aggregate plot (right panel) of CD38 expression on CD4+Glut1+ and CD4+Glut1− T cell from HIV+ subjects. (E) Representative histogram (left panel) and aggregate plot (right panel) of CCR5 expression on CD4+Glut1+ and CD4+Glut1− T cell from HIV+ subjects. 5 HIV− and 5 HIV+ subjects were analyzed for all surface markers. (F, G) Representative dot plots showing forward and side scatter properties of PBMCs from two HIV‐negative subject stimulated with 10 μg·mL−1 PHA plus 5 ng·mL−1 IL‐2 for 4 days (blue dots), or cultured without stimulation for the same amount of time (red dots) (left panel). (Right panels) Cells were labeled with CFSE on day 1 as described in Methods, and representative plots of CFSE‐labeled CD4+ T cells after 4 days of incubation with PHA plus IL‐2 (blue line) or unstimulated (red line). (H) The bar chart indicates the cumulative MFI of Glut1 relative to isotype control on CD4+ T cells within each corresponding peak (A–G) showing different rounds of CD4+ T cell replication, and peak G (red) showing CD4+ T cells from unstimulated PBMCs. Cumulative results are obtained from 4 to 5 peaks depending on the amount of cell division. The paired t‐test was used to measure significant differences within groups.

Fig 3: Effect of ethanol on CD4+ T cell glucose metabolism. (A) Representative FACS plots of GLUT1 expression in DIFF and DIFF + EtOH CD4+ T cells. (B) Ethanol increased CD4+ T cell GLUT1 expression. (C) Representative FACS histograms of 2-NBDG uptake in DIFF and DIFF + EtOH CD4+ T cells. (D) Ethanol increased glucose uptake within differentiated CD4+ T cells. (E) All experimental groups were analyzed using the Glycolytic Stress test. Ethanol increased glycolysis (F) and glycolytic capacity (G) within differentiated groups. MFI = mean fluorescent intensity, 2-NBDG = 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, ECAR = extracellular acidification rate, Naïve = undifferentiated and no ethanol treatment, EtOH = undifferentiated and ethanol-treated, DIFF = differentiated and no ethanol treatment, and DIFF + EtOH = differentiated and ethanol-treated. Data represents average values using CD4+ T cells from 6 donors expressed as mean ± SEM. Significant differences (p<0.05) were determined by repeated measures 2-way ANOVA. *p ≤ 0.05.

Fig 4: Gene expression changes after 4 (d4) and 9 days (d9) of culture under different 2D and 3D conditions. Selected genes having cell surface localized protein product and well characterized antibody available on the market: TMEM5A, SLC16A3 (MCT4), CEACAM5, SLC2A1 (GLUT1), and CA9 showed 16–32 times overexpression in 3D Cytodex3 and 3D Nutrisphere compared to 2D monolayer. Data are mean ± SD of three replicates.

Fig 5: Model illustrating metabolic exhaustion of CD4 + T cells in HIV-infected individuals. Activation of CD4 + T cells is maintained by increased cell surface Glut1 expression and glycolysis. We propose a model whereby CD4 + T cells have a low energy reserve and therefore, prolonged activation results in metabolic exhaustion and cell death. PPP, pentose phosphate pathway.