Fig 1: Complementary activating and inhibitory polarity in mitotic T cellsCD8+ T cells blasts from B16-OVA-challenged mice after 10 days were analyzed by confocal microscopy.(A) Left: LN cell with co-localized polarity of CD28 and PIP3. Center: quantitation of CD28 and PIP3 asymmetry using DNA as a reference for symmetry. Each line depicts values of a single interphase (black) and mitotic (red) cell. Right: frequency of CD28 and PIP3 polarity being on the same (black) or opposite (gray) side among pooled LN and tumor cells in interphase (I) and mitosis (M) .(B) Top: cells with PD-1 polarity opposite from polarity of CD3, CD28, and PIP3. Paired red arrowheads mark oppositely facing MTOCs or spindle poles of mitotic events. Bottom: quantitation of asymmetry in cells with polarity of an activating marker (; CD3, CD28, or PIP3) and PD-1 relative to the DNA signal. Lines with negative PD-1 values represent events with PD-1 polarity opposite from CD3 polarity.(C) Frequency of PD-1 polarity on the same (black) or opposite (gray) side as activating marker polarity (Activ; CD3, CD28, or PIP3) in I versus M.(D) Top: representative LN cells with opposite-sided CD73 and PIP3 polarity. Bottom: quantitation of CD73 and PIP3 asymmetry relative to the DNA signal.(E) Frequency of CD73 polarity on the same (black) or opposite (gray) side as PIP3 polarity in I versus M.(F) Cumulative frequency of inhibitory polarity (PD-1 or CD73) being on same or opposite side as activating polarity (CD3, CD28, or PIP3) in I versus M.Scale bars indicate 2.5 μm. Four replicate experiments with at least 5 mice in each experiment were performed. The number of cell events per group is indicated. Statistical comparison of inhibitory molecule and DNA polarity scores was stratified into those with inhibitory polarity on the same or opposite side as activating polarity. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple-comparisons tests (A, B, and D) and binomial test (C, E, and F): ns, non-significant; *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S1.