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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: J Immunol. 2022 Aug 5;209(5):864–873. doi: 10.4049/jimmunol.2200145

Figure 1. Activated TCD4+ and DCs both express antigen presentation machinery components and have endocytic protease activities but TCD4+ have weak internalization capabilities.

Figure 1.

(A) percent positive and (B) relative mean fluorescence intensity (MFI) of HLA-DR, CD86, HLA-DM, and invariant chain in uninfected TCD4+ and DCs. TCD4+ and DCs were cultured as described. Cells were then stained for flow cytometry. HLA-DR and CD86 positive populations were gated on live singlets. HLA-DM and invariant chain expression was determined by pre-gating HLA-DR and CD86 double positive cells. (C) Percent positive and (D) relative MFI of these markers in uninfected (UI) and WT HIV-1 infected (HIV+) TCD4+. Where indicated, activated TCD4+ were infected with 100 ng p24 HIV-1 3 days prior to staining. Activated TCD4+ and DCs lysates were then analyzed for (E) cathepsin D, (F) cathepsin L, and (G) cathepsin S activity using fluorometric cathepsin substrates. Lysates from 1 × 105 cells were analyzed per replicate. Measurements were taken after 30 mins at 37°C. Activated TCD4+ and DCs were then tested for their ability to internalize and proteolyze DQ-OVA. All values are background subtracted. (H) Percent DQ-OVA positive cells and (I) MFI in DQ-OVA+ cells. TCD4+ and DCs were cultured as previously described and incubated with DQ-OVA fluorescent substrate for 2 hours at 37°C. Uptake and proteolysis were measured by flow cytometry on live singlets. Gates were drawn based on no-substrate controls. Percent FITC-Dextran positive cells (J) and (K) MFI in FITC-Dextran+ cells. TCD4+ and DCs were cultured as previously described and incubated with FITC-Dextran fluorescent substrate for 2 hours at 37°C. Uptake and proteolysis were measured by flow cytometry on live singlets. Gates were drawn based on no-substrate controls. Each color represents a unique donor, as indicated in the figure legend. For G-K, each dot represents a technical replicate. Representative of 3 independent experiments with the exception of L and M, which are representative of 2 independent experiments. For A-F and L-M, data were analyzed with an unpaired t test. For G-K, data were analyzed with a One-way ANOVA. Bars represent mean ± SD. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.