Tonsil cells were exposed to KSHV and analyzed by MIFC for expression of LANA, IgM, and CD27 at 72 hpi. (A) Representative images of cells labeled with antibodies to LANA, IgM, and CD27. (B) Left panel: Histogram showing representative gating for IgM+ cells. Right panel: Percentage of IgM– or IgM+ cells expressing LANA dots, from 5 donors (individual symbols). (C) Left panel: Histogram showing, among IgM+ cells (gated as in B), levels of IgM expression on LANA– (gray line) versus LANA+ (black line) cells. Right panel: Among IgM+ cells, fold change in IgM expression (MFI) of individual LANA+ groups (1–2, 3–4, ≥5 dots/cell) compared with LANA– (0 dots) cells. Values represent mean ± SEM from 5 separate tonsil donors. P value reflects significance of Spearman correlation, which was 0.797. (D) Left panel: Representative histogram showing CD27 expression on LANA–IgM+ (gray filled) and LANA+IgM+ (black line) cells. Vertical line indicates threshold above which CD27 signal becomes visible by MIFC. Above left: Examples of cells on either side of threshold. Right panel: Percentage of IgM+LANA– or IgM+LANA+ cells expressing CD27 (5 donors). (E) Scatter plot showing nuclear and cytoplasmic areas for LANA– cells (gray dots) and LANA+ cells (green circles). (F) Mean N/C ratios for uninfected (0 dots) and cells with low (1–2 dots), moderate (3–4 dots), and high (≥5 dots/cell) viral load. Data represent mean ± SEM from 4 of 6 KSHV-exposed tonsils. P value reflects significance of Spearman correlation, which was 0.675.