Figure 7.

CTLs Trigger Ca²⁺ Fluxes in Virus-Infected Cells and Cooperate during Killing

(A) One day after infection with MCMV-expressing Ca²⁺ sensor GCaMP6s (MCMV-3D-ΔvRAP-Ca), two-photon microscopy was used to record GCaMP6s intensity (green) in virus-infected cells (red).

(B) Kinetics of GCaMP6s intensity in an infected cell imaged at 0.2 Hz.

(C) Ca²⁺ flux (defined as GCaMP6s^bright event) duration in virus-infected cells in the absence of specific CTLs (dots represent cells, and red bars represent IQRs; n = 98 infected cells from three mice).

(D) Kinetics of Ca²⁺ fluxes in one infected cell imaged for 10 min at 0.07 Hz.

(E) In lymph nodes with MVA-OVA-primed CTLs present, a CFP⁺ OT-I CTL (blue; dotted line) contacted a MCMV-3D-ΔvRAP-Ca-infected cell (long flux defined as GCaMP6s^bright event lasting >30 s).

(F) Kinetics of a long-lasting Ca²⁺ flux of an infected cell imaged for 10 min at 0.05 Hz. The green line represents the locally weighted scatterplot smoothing curve.

(G) Duration of Ca²⁺ fluxes of infected cells that were not contacted, were contacted but stayed intact, or were contacted and killed. Dots represent cells, and red bars represent IQRs. A Kruskal-Wallis test was used for comparing multiple groups. Data were pooled from four experiments from six different mice for a total of 307 flux events analyzed. ^∗∗p < 0.01; ^∗∗∗p < 0.001; ns, not significant.

(H) Time interval between CTL contact and subsequent long-lasting Ca²⁺ flux (n = 79 events from four experiments analyzed; red bars represent IQRs).

(I) Percentages of CTL contacts that were followed by a long-lasting Ca²⁺ flux (n = 128 CTLs). Data were pooled from four experiments.

(J) Percentages of different numbers of long-lasting Ca²⁺ fluxes that followed a CTL contact event (n = 77 CTLs). Data were pooled from four experiments.

(K) Percentage of long-lasting Ca²⁺ fluxes that followed contacts of Prf^−/− CTLs (n = 51 contacts pooled from three experiments).

(L) Probability of target-cell death for infected cells contacted by 0–14 CTLs. Hypothetical values for the no-cooperativity null hypothesis (open dots) and observed data (red squares) are provided. In total, 660 infected cells were analyzed, and data were pooled from >12 independent experiments.

(M) p values for comparing data derived from the null hypothesis and observed data (0.05 significance level indicated by dotted line); see also Figures S5, S6, and S7.