Figure 4.

PICALM KO causes increased cell proliferation and deregulates activation and PD-1 pathways

(A) SupT1 PICALM KO T cells were transfected with a ‘rescue’ plasmid to recover PICALM expression. SupT1 WT, PICALM KO T cells and KO PICALM expressing T cells were evaluated by flow cytometry for expression of Ki67, demonstrating that PICALM cells have increased propensity to proliferate.

(B and C) WT and PICALM KO T cells were infected with WT NL4-3 virus (MOI = 5) for 48 h. Cells were analyzed for cell surface expression of PD-1 and CD69 expression flow cytometry, demonstrating that PICALM KO reduces expression of PD-1, and increases CD69, independently of virus infection.

(D) Flow cytometry was used to assess cell surface expression of PD-1 of PICALM KO cells using two different antibodies, validating that PICALM KO decreases PD-1 expression.

(E) Flow cytometry was used to measure expression of INF-γ of PICALM KO, demonstrating that PICALM KO increases INF-γ expression.

(F–I) WT and PICALM KO T cells were infected with WT NL4-3 virus (MOI = 5) for 48 h. Cells were analyzed for both cell surface expression and overall cellular expression of numerous immunological biomarkers using a cell permeabilization staining flow cytometry protocol. Bar graphs demonstrate that PICALM modulated expression of both PD-1 (F), CD25 (G), and TLR4 (H) is cell surface dependent. (I) CD4 was also analyzed by this assay, demonstrating that both cell surface and whole cell CD4 are downregulated by HIV-1 infection, but that CD4 levels are unaffected by PICALM KO in either case. (A) Data represents the percentage (%) of cells positive for Ki67. (B-I) Data were calculated from MFI (mean fluorescence intensity) of respective marker and relative % is calculated by setting wildtype uninfected (WT UI) values to 100%. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; 1-way ANOVA, Tukey post-test.