FIG 7.
Comparative analysis of MT-C34-expressing CD4 lymphocytes generated via knock-in or lentiviral transduction. (A) A typical live and dead CD4 lymphocyte distribution on the light scatterplots measured at day 3 postelectroporation or -lentiviral (LV) transduction. (B and C) The levels of MT-C34 (B) and CD25 (C) cell surface expression measured at day 5 posttransfection or transduction. The flow cytometry dot plots recorded for donor 2 and the MFI values calculated for both donors are shown on the left and right, respectively. (D) HIV-1 single-cycle coculture infection was set up by mixing transfected HIV-1 effector Raji cells with MT-C34-positive or -negative CD4 lymphocytes or Raji cells as a control at a 3:1 ratio. The levels of inLuc transduction measured 3 days later were adjusted to the number of target cells and presented separately for each donor. The average values from two donors were calculated, and the fold reductions in HIV-1 infectivity for MT-C34-negative and -positive samples are indicated. The dashed line indicates the background level of luciferase activity. The values obtained for donors 1 and 2 are shown by orange and blue circles. KIRNP stands for MT-C34 knock-in generated using Cas9 RNP. LV, lentiviral transduction as a method of MT-C34 peptide delivery. (+) and (−), isolation of MT-C34-positive and -negative cells. Differences are significant at P < 0.05 (*) by Student's t test. Representative flow cytometry images are shown in panels A to C.