Target cell motility directly impacts CTL function.

<p>(<b>A–C</b>) CD4⁺ target cells (controls or pulsed with KK10 gag peptide) were spun to the bottom of collagen matrices prior to gelation to allow binding to the underlying glass substrate, which was coated with anti-CD4 antibody and ICAM or ICAM alone. E501 CTLs were added to the matrix (E:T ratio 1∶2) and target/CTL dynamics were recorded by videomicroscopy for 10 hr. (<b>A</b>) Wind-rose plots of target cell migration over a period of 20 min in the absence or presence of immobilizing anti-CD4. (<b>B</b>) Target cell death was assessed by sytox fluorescence after 10 hr. (<b>C</b>) Engagement histories were recorded for motile or immobilized targets that were killed. (<b>D, E</b>) A14 CTLs were loaded with FURA-2 AM and Ca²⁺ signaling was monitored for CTLs engaging peptide-pulsed CD4⁺ targets (2 nM SL9, 1 hr time-lapse). (<b>D</b>) Shown are representative Ca²⁺ traces (blue) and instantaneous cell velocities (CTL in red, target in black) over time on the x-axis. The period of CTL-target engagement is denoted on the time axis (live target in pink, killed in gray). (<b>E</b>) The presence or absence of Ca²⁺ signals was scored for CTL-target engagements concluding with target death (<i>n</i> = 89), a failed tether (<i>n</i> = 112), or a brush (<i>n</i> = 95). Data pooled from 6 independent experiments. Bars indicate mean ± SEM.</p

Peptide-pulsed targets elicit CD8⁺ T cell engagement dynamics similar to those observed for HIV-infected targets.

<p>A14 CD8⁺ T cells embedded with primary CD4⁺ T cell targets (pulsed with the indicated doses of peptide) within collagen gels were imaged for 10 hrs and analyzed for CTL-target engagement dynamics (E:T ratio 1∶2). (<b>A</b>) CTL killing activity (% sytox⁺ targets) vs. time as a function of antigen pulsing concentration. Background cell death in the absence of added antigen primarily reflected a low level of spontaneous target cell apoptosis early in the co-cultures. (<b>B</b>) Frequency of each type of CTL-target engagement during the first 30 minutes of co-culture as a function of antigen pulse concentration. (<b>C</b>) Time elapsed from initial contact until blebbing and/or permeabilization and total duration of CTL-target contact is shown for engagements resulting in target death (targets pulsed with 20 nM SL9 peptide). (<b>D</b>) Durations of characteristic CTL-target engagements determined for targets pulsed with 20 nM SL9 peptide.</p

<i>In situ</i> reporters enabling continuous videomicroscopy of CTLs and primary CD4⁺ target cells in ECM.

<p>(<b>A</b>) Confocal images of CTL clone A14 labeled with CTXB (red) migrating through 3D ECM (left: brightfield/CTXB fluorescence overlay, right: reflectance image of collagen type I fibers). Scale bar 20 µm. (<b>B</b>) Mean velocities for individual cells tracked for one hour in collagen. HIV-specific CTLs (primary or clones), primary uninfected CD4⁺ T cells or HIV-infected CD4⁺ T cells were cultured independently within ECM. CD4⁺ T cells were infected with HIV-1 NL4-3-GFP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087873#pone.0087873-Brown1" target="_blank">[61]</a> and flow-sorted to >95% GFP⁺ populations on day 3 post-infection. Shown is one representative of 3 independent experiments. Bars indicate mean ± SEM. (<b>C</b>) A14 CTLs were co-cultured in ECM with HLA-matched primary CD4 targets (pulsed with 0 or 200 nM SL9 peptide). Antigen+/− samples were imaged in parallel at 1 min intervals for 10 hr with control samples imaged only at time 0 and 10 hr. Permeabilized target cells were assessed by CTXB⁻ sytox⁺ cell counts (left panel) and live CD8⁺ T cells were assessed by CTXB⁺ sytox⁻ cell counts (right panel). Shown is 1 representative of 4 independent experiments.</p

CTLs exhibit dynamic engagements with HIV-infected CD4⁺ target cells.

<p>(<b>A–C</b>) NL4-3-GFP HIV-infected CD4⁺ T cells (>95% GFP⁺) were imaged in collagen with E501 CTLs (E:T ratio 1∶2, 10 hr timelapse). (<b>A</b>) Contact types were characterized as (<b>i</b>) Direct hit kills, (<b>ii</b>) successful tethers, (<b>iii</b>) Failed tethers, and (<b>iv</b>) brushes. Shown are representative velocity traces (CTL in red, target in black) and the period of CTL-target engagement (live target in pink, morphologically dead target in gray, permeabilized target in green) vs. time for individual engagements of CTLs with infected targets. (<b>B</b>) Time elapsed from initial contact until blebbing and/or permeabilization and total duration of CTL-target contact is shown for engagements resulting in target death. (<b>C</b>) The number of escapes were enumerated for individual HIV-infected targets (n = 17 target cells analyzed). Engagements resulting in target death (red) or target escape (white) are indicated. (<b>D</b>) JR-CSF HIV-infected CD4⁺ T cells were cultured in liquid media or in ECM in the absence or presence of CTL clone A14 or E501 (E:T ratio 1∶1) and HIV replication relative to infected cells alone was assessed by p24 ELISA after 2 days. Data are from one representative of 3 independent experiments. Bars indicate mean ± SEM.</p

CTLs rapidly transition from killing to sustained non-lytic effector secretion during prolonged arrest.

<p>A14 CTLs and peptide-pulsed CD4⁺ target cells (20 nM SL9) were co-cultured in collagen (E:T ratio 1∶2) and supernatants were harvested after the indicated time periods. Concentrations of secreted cytokines/chemokines were analyzed using Flex Set bead-based ELISAs (BD Biosciences). Each data point reflects a unique set of triplicate wells for each timepoint or condition. (<b>A</b>) Kinetics of cytokine/chemokine secretion were determined for CTL-target co-cultures or for CTLs incubated alone over a 24 hour time period. (<b>B</b>) Target cells were pulsed with SL9 or double mutant SL9 peptide and co-cultured in the presence or absence of the TCR signaling inhibitor dasatinib added after 1 hr or 5 hr of co-culture; secreted cytokine concentrations were assessed as in (A). **** p<0.0001. Significance of data was determined by a 2 way ANOVA followed by a Bonferroni post test. Data shown are from 1 representative of 3 independent experiments. Bars indicate mean ± SEM.</p

CTL exhibit a prolonged TCR-dependent, CTL-intrinsic migration arrest after successfully engaging and killing an initial target.

<p>(<b>A</b>) A14 CTLs were co-cultured in collagen with CD4⁺ target cells pulsed with SL9 peptide (E:T ratio 1∶2) and total engagement times for CTL-target encounters ending in target death were recorded. Data shown from 1 representative of 5 independent experiments. Bars indicate mean ± SEM. (<b>B</b>) The duration of E501 CTL engagements with 15 µm beads presenting recombinant B27-KK10 peptide-MHC complexes at a density of 20 pMHC/µm² or control beads lacking pMHC in collagen was quantified by videomicroscopy. Data are from 1 representative of 4 independent experiments. Bars indicate mean ± SEM. (<b>C</b>) A14 CTLs were imaged in collagen with CD4⁺ T-cells pulsed with the indicated doses of SL9 peptide in collagen (E:T ratio 1∶2, 10 hr timelapse). Outcomes for each CTL engaging its first target or subsequent targets are expressed as % of first CTL encounters or % of subsequent encounters (<i>n</i> = 185 (0.1 nM SL9), 147 (2 nM SL9), and 308 (20 nM SL9) engagements analyzed). Data are pooled from 3 independent experiments.</p

High antigen sensitivity and adhesion receptors CD58 and LFA-1 are required for efficient killing of motile targets by individual CD8⁺ T cells.

<p>CD8⁺ T cell clones were co-cultured in ECM with HLA-matched CD4⁺ targets (E:T ratio 1∶2). (<b>A</b>) Functional avidities of A14 and E501 CTLs engaging targets in collagen were determined by ⁵¹Cr release assays, by determining EC₅₀s (antigen pulsing concentration required for 50% of max target death, for SL9 peptide and SL9 variants or KK10 peptide, respectively). (<b>B</b>) CTL efficiency was observed at the single-cell level (% of individual CTL successfully engaging the first motile target encountered). Data pooled from 3–5 independent experiments, <i>n</i> for each condition within each experiment was 10–30 CTL. Bars indicate mean ± SEM. (C–D) A14 CTLs were co-cultured with HLA-matched CD4⁺ targets (pulsed with 2 nM SL9) within ECM in the presence of the indicated adhesion receptor antagonists (10 µg/mL neutralizing antibody or 50 nM inhibitor) or isotype and vehicle controls. (<b>C</b>) CTL killing activity was measured by ⁵¹Cr release from targets after 6 hr of co-culture within ECM. Data pooled from 3 independent experiments. Bars indicate mean ± SEM. (<b>D</b>) Frequencies of characteristic A14 CTL-target engagements during the first 30 minutes of videomicroscopy.</p