Fig 2: HIV Infection Induces the Expression of SLeX and CLA on the Surface of Primary CD4+ T Cells In Vitro(A) Isolated CD4+ T cells from three HIV-negative donors were infected with either the HIV 89.6 virus (at a multiplicity of infection [MOI] of 6) or the HIV DH12 virus (at MOI of 10) by spinoculation in the presence of CD44 MicroBeads. Four days later, intracellular p24 expression and cell-surface expression of SLeX were determined by flow cytometry.(B) Isolated CD4+ T cells from three HIV-negative donors were depleted of CLA+ cells. CLA-depleted cells were infected with either HIV 89.6 or HIV DH12 by spinoculation in the presence of CD44 MicroBeads. Four days later, intracellular p24 expression and cell-surface expression of CLA were determined by flow cytometry. (A and B) Top panels show the percentage of SLeX or CLA on total, p24+, and p24-negative cells, as well as controls (cells treated with CD44 MicroBeads alone), for each HIV strain. Bottom panels are representative flow cytometry plots of dually stained CD4+ T cells for HIV p24 protein and SLeX or CLA. Mean and standard error of the mean (SEM) are presented. Paired t tests were used for statistical analysis.(C) H9 cell line was infected with either HIV 89.6 (MOI of 6) or NL4–3 (MOI of 0.1) in the presence of CD44 MicroBeads. Protein expression of several glycosyltransferases involved in SLeX and CLA production (FUT6, FUT7, and B4GALT5), as well as HIV p24, was measured by western blotting. Diagrams show locations of glycosidic bonds catalyzed by indicated glycosyltransferases.

Fig 3: Example of Analysis of Activation Markers in CLA+/hi/low CD4+ T CellsRepresentative dot plots gated on total CD4+ T cells as shown in Figure 5, stained on cryopreserved PBMC from an HIV-infected individual under antiretroviral treatment (using the antibody cocktail detailed in Table 4).(A) Gating on HLA-DR+ CD4+ T cells, followed by selection of subsets based on expression of CLA. Number inside the gate indicates the percentage of HLA-DR+ CD4+ T cells.(B) Gating of subsets based on expression of CLA, followed by an overlay plot showing the selection of HLA-DR+ cells within each subset. Corresponding frequencies are shown to the right of the plots.(C) FMO used to determine the HLA-DR+ gate.(D) Example of biological gating based on CD4+ T cell co-expression of CD38 and HLA-DR.(E) Example of biological gating using expression of CD27 and HLA-DR, and naive cells as a reference to determine positive and negative populations. Total memory CD4+ T cells were selected as shown in Figure 6.

Fig 4: Example of Gating Strategy for the Characterization of Th Subsets in Memory CD4+ T CellsRepresentative example of analyses in a cryopreserved PBMC sample. Memory CD4+ T cells are first selected by excluding naive cells (CCR7+ CD45RA+ CD4+ T cells). Inside memory CD4+ T cells, SLeX+ were selected from Stain 1, using the antibody cocktail described on Table 3. CLA+ cells (black gate), CLAhi (teal gate), and CLAlow (orange gate) were selected from Stain 2, using the antibody cocktail described in Table 4. cTfh cells are identified using PD-1 and CXCR5. CD127low CD25+ cells were selected within non-cTfh cells, and Foxp3+ cells were then selected to identify Tregs. Non-cTfh/Tregs cells were selected based on CXCR5 and CCR6 expression. Th1 and Th2 cells were identified within the CXCR5- CCR6- gate, while Th17 and Th17+1 were characterized ins the CXCR5- CCR6+ gate using CXCR3. The frequency of CXCR3+ Tfh can also be measured with this panel.

Fig 5: CLAhigh CD4+ T Cells Cluster Distinctly from CLAlow and CLAnegative CD4+ T Cells(A) Left, heatmap showing the frequency of all measured phenotypic markers in CLAhigh, CLAlow, and CLA- cells. Right, PCA showing the distribution of CLAhigh, CLAlow, and CLA- cells subsets.(B) Memory subset distribution of CLAhigh cells in HIV-, HIV+ ART-treated, and HIV+ viremic individuals.(C) Frequency of CLAhigh cells within each memory CD4+ T cell subset in the analyzed groups. Lines and error bars represent the median and IQR. All statistical comparisons were performed using two-tailed Wilcoxon rank tests.(D) Heatmaps showing the statistical difference in the frequency of all measured phenotypic markers CLAhigh, CLAlow, and CLA- cells.All data were analyzed using a Friedman test (paired, non-parametric). n = 6 for HIV-, 7 for HIV+ ART-suppressed individuals, and 8 for HIV+ viremic individuals. *p < 0.05, **p < 0.01, ***p < 0.001.