Figure 5.
Ectopically expressed TPST2 in primary B cells does not improve eCD4-Ig-KiHR HIV-1 neutralization functionality
(A) TPST2-P2A-eCD4-Ig LV schematic with the constitutive (MND-M) promoter. The LV elements are as described in Figure 1B. (B) Representative anti-sulfotyrosine non-reducing western blot analysis from supernatants of HEK293T cells transfected with the pM-TPST2-eCD4-Ig-KiHR or pM-rGFP-eCD4-Ig-KiHR LV plasmids. Supernatants were harvested 4 days post transfection and evaluated to verify the presence of eCD4-Ig-KiHR using an anti-CD4 ELISA assay as described in materials and methods. Next, supernatants containing equal amounts of eCD4-Ig-KiHR (20 mg) were analyzed by SDS-PAGE and western blot analysis using anti-sulfotyrosine antibody to detect sulfonylation of eCD4-Ig-KiHR. (C) Stable LV transduction of primary B cells with M-TPST2-eCD4-Ig-KiHR LV did not enhance the HIV-1 neutralization functionality of eCD4-Ig-KiHR. B cell cultures were established as described in Figure 4A, and on day 10 culture supernatants were collected and evaluated for eCD4-Ig-KiHR amounts using anti-gp140 ELISA. HIV-1 neutralization assays were performed as described in Figure 4C and materials and methods, using equal amounts of eCD4-Ig-KiHR-containing supernatants to evaluate the effect of ectopically expressed TPST2 on eCD4-Ig-KiHR HIV-1 neutralization functionality. Purified eCD4-Ig was used as a positive control, and supernatant from mock untransduced B cells was used as a negative control. Blue horizontal line marks IC50, and graph points show range and average of duplicates. Note overlapping IC50S intersect (blue line)for all HIV-1 isolates and all are within the assay variance. IC50s were calculated from six dilution points of each supernatant. (D) TPST2 is highly expressed in B cells. RNA sequencing was performed on CD138+ cells enriched from human plasma cell cultures. Raw counts were averaged for the top 500 expressed genes (n = 4 donors), and the results are plotted. Red arrow identifies TPST2 gene location.