High Avidity CD8+ T Cells Efficiently Eliminate Motile HIV-Infected Targets and Execute a Locally Focused Program of Anti-Viral Function

The dissemination of HIV from an initial site of infection is facilitated by motile HIV-infected CD4+ T-cells. However, the impact of infected target cell migration on antigen recognition by HIV-specific CD8+ T-cells is unclear. Using a 3D in vitro model of tissue, we visualized dynamic interactions between HIV-infected or peptide-pulsed CD4+ T-cells and HIV-specific CD8+ T-cells. CTLs engaged motile HIV-infected targets, but ∼50% of targets broke contact and escaped. In contrast, immobilized target cells were readily killed, indicating target motility directly inhibits CD8+ T-cell function. Strong calcium signals occurred in CTLs killing a motile target but calcium signaling was weak or absent in CTLs which permitted target escape. Neutralization of adhesion receptors LFA-1 and CD58 inhibited CD8+ T-cell function within the 3D matrix, demonstrating that efficient motile target lysis as dependent on adhesive engagement of targets. Antigen sensitivity (a convolution of antigen density, TCR avidity and CD8 coreceptor binding) is also critical for target recognition. We modulated this parameter (known as functional avidity but referred to here as “avidity” for the sake of simplicity) by exploiting common HIV escape mutations and measured their impact on CTL function at the single-cell level. Targets pulsed with low avidity mutant antigens frequently escaped while CTLs killed targets bearing high avidity antigen with near-perfect efficiency. CTLs engaged, arrested, and killed an initial target bearing high avidity antigen within minutes, but serial killing was surprisingly rare. CD8 cells remained committed to their initial dead target for hours, accumulating TCR signals that sustained secretion of soluble antiviral factors. These data indicate that high-avidity CD8+ T-cells execute an antiviral program in the precise location where antigen has been sensed: CTL effector functions are spatiotemporally coordinated with an early lytic phase followed by a sustained stationary secretory phase to control local viral infection

Mutational sequencing for accurate count and long-range assembly

ABSTRACT We introduce a new protocol, mutational sequencing or muSeq, which randomly deaminates unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling a 135,000 fragment PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone

Precision measurement of cis-regulatory energetics in living cells

Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs). However, measuring TF-TF interactions that occur at individual cis-regulatory sequences remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. Our strategy uses reporter assays performed on strategically designed cis-regulatory sequences, together with a biophysical modeling approach we call "expression manifolds". We applied this strategy in Escherichia coli to interactions between two paradigmatic TFs: CRP and RNA polymerase (RNAP). Doing so, we consistently obtain measurements precise to ~0.1 kcal/mol. Unexpectedly, CRP-RNAP interactions are seen to deviate in multiple ways from the prior literature. Moreover, the well-known RNAP binding motif is found to be a surprisingly unreliable predictor of RNAP-DNA binding energy. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should thus be highly scalable and broadly applicable

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

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

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