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

Cellular Barcodes for Efficiently Profiling Single-Cell Secretory Responses by Microengraving

We present a method that uses fluorescent cellular barcodes to increase the number of unique samples that can be analyzed simultaneously by microengraving, a nanowell array-based technique for quantifying the secretory responses of thousands of single cells in parallel. Using n different fluorescent dyes to generate 2n unique cellular barcodes, we achieved a 2n-fold reduction in the number of arrays and quantity of reagents required per sample. The utility of this approach was demonstrated in three applications of interest in clinical and experimental immunology. Using barcoded human peripheral blood mononuclear cells and T cells, we constructed dose–response curves, profiled the secretory behavior of cells treated with mechanistically distinct stimuli, and tracked the secretory behaviors of different lineages of CD4+ T helper cells. In addition to increasing the number of samples analyzed by generating secretory profiles of single cells from multiple populations in a time- and reagent-efficient manner, we expect that cellular barcoding in combination with microengraving will facilitate unique experimental opportunities for quantitatively analyzing interactions among heterogeneous cells isolated in small groups (2–5 cells).Howard Hughes Medical Institute (Investigator)National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1R56AI104274)National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1U19AI089992)National Institute of Allergy and Infectious Diseases (U.S.) (Grant 5U01AI068618)National Science Foundation (U.S.) (Fellowship)Massachusetts Institute of Technology (Collamore-Rogers Fellowship)Ragon Institute of MGH, MIT and HarvardW. M. Keck Foundatio

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

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

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