Design and validation of a single-cell assay for antigen-specific cytolysis.
We designed an assay to observe the cytolytic activity of individual CD8+ T effector cells encountering discrete numbers of peptide-loaded APCs as targets. The approach uses a dense, elastomeric array of wells with subnanoliter volumes (125 pl each) to execute thousands of independent single-cell cytolytic assays in parallel by coincubating T cells with APCs (Figure A). On-chip, live-cell cytometry using automated microscopy determines the occupancy of each of the approximately 85,000 wells in the array, and inclusion of a fluorescent, membrane-impermeant nucleic acid stain (SYTOX) in the media during incubation allows the dynamic assessment of cell death in permeabilized cells, since those that die become fluorescent (green).
High-throughput, single-cell assay for cytolysis.
To validate the assay, we deposited a CD8+
T cell clone specific for the HIV-1 p24 Gag epitope KK10 (E501), restricted by HLA-B*27, into the wells of the array. Application of 300 μl containing 1.5 × 105
cells to the array yielded a distribution of approximately 1 cell per well. To minimize perturbations to the functional activity of the T cells, we did not label these cells at the time of deposition. For APCs, we used HLA-matched, EBV-transformed B cells, because as a population, they exhibited a consistent phenotype (expressing CD11c, CD70, and CD80, but not CD11b, CD83, or CD86) and thus also provided consistency in antigen presentation to facilitate comparisons among T cells in different microwells on the array and among populations of T cells derived from different subjects. (Such lines can also be generated in clinical studies to allow autologous antigen presentation; ref. 22
.) The B cells were labeled with a cytosolic dye (CellTracker Red) to identify metabolically active live cells, pulsed for 60 minutes with the cognate CTL epitope targeted by this clone, and then washed. A suspension of these cells was subsequently loaded onto the array of wells containing the effector cells. This two-step process for loading wells with both types of cells typically yielded 3 × 103
to 6 × 103
microwells containing exactly one effector and one target cell, with approximately 104
wells containing at least one effector and a single target (Supplemental Figure 1; supplemental material available online with this article; doi:
). The APCs were pulsed with a high dose of the peptide to promote uniform presentation by each individual APC, but excess antigen was removed by washing the cells prior to their deposition onto the array. In this way, the number of target cells in each well defined the maximum dose of antigen to which co-loaded T cells could be exposed.
After the array was loaded with both target and effector cells, it was immersed in media containing SYTOX and imaged to determine cell viability. Wells containing dead target cells at the start of the assay (SYTOX-positive) were excluded from further analysis. Arrays were incubated for 4 hours, followed by in situ labeling of cells with a live-cell stain (calcein violet), and imaged again. These images were used to determine the number of viable T cells (blue), plus the numbers of viable (red) and dead (red + green) targets, in each well.
Automated quantitative analysis of more than 5,000 independent T cell–APC interactions per experiment was performed to determine the specificity of lysis by E501 cells incubated with target APCs with or without the cognate KK10 epitope (Figure , B and C). Wells containing E501 effector cells coincubated with KK10-loaded APCs showed increased numbers of dead target cells over 4 hours (29%–47%). In the absence of this peptide, the number of dead APCs observed in wells containing E501 cells after 4 hours was 2%–3%, which was comparable to the typical frequency of spontaneous death observed in wells containing only APCs (Supplemental Figure 2). Time-lapse video microscopy on loaded arrays confirmed that the effector cells were highly mobile in the wells during the period of incubation, typically engaging target APCs within an hour of loading and subsequently permeabilizing the membranes of cells pulsed with matched peptide (Supplemental Video 1). These single-cell data were consistent with the specific lysis observed after 4 hours using the same populations of effector and target cells in a 51Cr release assay (27%–45%, Figure D).
Effector-mediated lysis was confirmed to be specific to the cognate epitope in a crossover experiment. B cells were pulsed with either KK10 or a different Gag epitope, SL9 (1 μg/ml), and then loaded into microwells containing CD8+ T cell clones specific to either KK10 (clone E501) or SL9 (clone A14) (minimum n = 2,924 per experiment). Effector-mediated lysis was 29%–44% when matched peptides were used (E501 clones with KK10-pulsed targets; A14 clones with SL9-pulsed targets), but less than 1% when T cell clones were coincubated with B cells loaded with irrelevant peptides (E501 clones with SL9-pulsed targets; A14 clones with KK10-pulsed targets). Together, these experiments using T cell clones demonstrated the feasibility of measuring HIV-specific cytolytic activity by on-chip cytometry for many thousands of independent events in parallel in spatially defined arrays of microwells.
Detection of HIV-specific cytotoxic effector T cells from clinical samples.
These experiments with T cell clones suggested that it should be possible to detect HIV-specific CD8+ T cells from clinical samples based on their cytolytic activity. To examine this hypothesis, we identified a subject who controls HIV spontaneously and has a robust response to the same B*27-restricted Gag KK10 epitope targeted by the clone. Approximately 7%–8% of circulating CD8+ T cells from peripheral blood were KK10 specific by MHC class I tetramer staining (Figure A). Analysis by flow cytometry indicated that the population of KK10-specific CD8+ T cells comprised a range of differentiated states (predominantly TEM, TEMRA, and TCM, where EM indicates effector memory; EMRA, effector memory RA; and CM, central memory) (Supplemental Figure 3). This population of cells reduced viral replication of an HIV laboratory strain in vitro using a viral suppression assay measuring released p24 (Figure B), with a smaller percentage of antigen-dependent responses detected by IFN-γ ELISpot (2,277 SFU/106 PBMCs).
Ex vivo screening and recovery of HIV-specific cytotoxic effectors from clinical samples.
For direct assessment of cytolytic activity, CD8+ T cells were isolated from previously frozen PBMCs by negative enrichment with antibody-coated magnetic beads and coincubated with labeled, peptide-pulsed (B*27-KK10, 5 μg/ml), HLA-matched B cell targets in arrays of microwells in the same manner as described above. After incubation for 6 hours, target cell death was identified based on SYTOX staining (Figure C). Antigen-dependent cell lysis was reproducibly detected in wells containing both T cells and target cells with cognate peptide (136 of 3,029 events; 4.5%), whereas the number of target cells spontaneously dying on-chip was only 66 of 2,485 (2.7%). The small antigen-dependent difference measured was statistically significant (Fisher’s exact test, P = 0.0003) and reproducible in 4 independent replicates of the experiment. The number of dead cells observed when target cells presented an irrelevant melanoma epitope was not different from spontaneous deaths (108 of 3,864; 2.8%, P = 0.90).
To verify further the antigenic specificity of the identified CTLs, individual cells that mediated killing in the assay were then retrieved by micromanipulation and seeded into 96-well plates containing irradiated HIV-uninfected PBMCs for clonal expansion (23
). Although not all of these cells could be propagated in culture, 3 of 3 successfully expanded cytolytic CD8+
T cells from the microwell array were highly specific for the KK10 epitope, as determined by MHC class I tetramer staining (Figure D). CD8+
T cells randomly selected from wells without targets proliferated robustly, but none were antigen specific, as expected given the frequency of antigen-specific cells in the CD8+
T cell population. The limited efficiency of expansion among the cytolytic effector T cells likely indicates that the majority of cytolytic events measured result either from cells with a terminally differentiated phenotype that cannot expand or from cells that subsequently die due to activation-induced cell death (24
Sequence analysis of the T cell receptors from all three recovered antigen-specific clones showed distinct differences in Vα and Vβ chains (Supplemental Table 1). These clones also exhibited robust antiviral activity (10,000- to 100,000-fold reduction in measured p24 on day 7) in a viral suppression assay that measured their ability to inhibit replication of an HIV-1 laboratory strain (JRCSF) in activated, HLA-matched CD4+ T cells (Figure E). This result was similar to the antiviral activity that had been observed using tetramer-based sorting of KK10-specific CD8+ T cells from peripheral blood of this person (Figure B). These results show that the microwell assay can detect individual, antigen-specific cytolytic effector cells within peripheral blood and that a diversity of clonotypes is primed for rapid cytolysis within the subset of T cells that label with MHC I tetramers.
Integrated measure of short-term cytokine secretion and immediate cytotoxicity.
The production of IFN-γ has been used as an assumed surrogate for HIV-specific cytolytic activity during the progression of disease and to assess the immunogenicity of candidate vaccines. In the absence of a direct assay to measure both functions (cytolysis and secretion), it has remained unclear to what extent cells exhibit both responses concurrently when encountering an APC. We have previously shown that the elastomeric array of microwells used here can also detect the frequencies and rates of secretion of multiple cytokines by a process called microengraving (26
). According to this method, a glass slide coated with antibodies specific for cytokines of interest is used to seal the microwells, and thereby confines the cells to the subnanoliter volumes of the microwells. After a short incubation (1–2 hours), the glass is removed to yield an array of cytokines registered to the array of microwells. Using this method, we have demonstrated, and confirmed with quantitative models, the ability to measure the secretion of IFN-γ, IL-2, and TNF-α simultaneously from polyclonally activated, primary T cells ex vivo with sensitivities 10- to 100-fold greater than ELISpot and surface-based capture (27
We integrated the single-cell cytolytic assay described here with microengraving to measure the relationship between cytokine release and cell killing by individual antigen-specific CD8+ T cells when encountering one or a small number of APCs (Figure A). To first validate this integrated assay, fluorescently labeled, peptide-pulsed (B*27-KK10, 5 μg/ml) B cell targets were coincubated with unlabeled E501 effectors in arrays of microwells, in the presence of costimulatory antibodies (αCD28 and αCD49d) to further minimize heterogeneity in antigen presentation among the APCs. After incubation for 4 hours, effector-mediated lysis was identified by image-based cytometry. The array of microwells was then rinsed with serum-free medium and placed in contact with a glass slide bearing antibodies specific for IFN-γ, IL-2, and TNF-α. Following incubation for an additional 2 hours at 37°C, the glass slide was removed, labeled with fluorescent antibodies, and imaged to determine wells that contained cells secreting cytokines (Figure B).
Integrated single-cell analysis of immediate cytotoxicity and short-term cytokine secretion from T cell clones.
The majority of the clonal E501 cells exhibited lytic activity and secreted both IFN-γ and IL-2, but not TNF-α (Figure C). For various ratios of effectors to targets (E/T = 1:1–1:6), 67%–76% of effectors secreted both IFN-γ and IL-2 and lysed their target-cells; 3% secreted only IL-2 and induced lysis. Target cells lacking antigen and coincubated with E501 effectors showed 1%–2% spontaneous lysis, with no detectable cytokine secretion, confirming that the release of these cytokines depends on antigen-specific recognition. This combined assay, therefore, enables direct monitoring of both antigen-induced cytotoxicity and secretion of cytokines for individual effector cells, and shows that an IL-2–dependent HIV-specific CTL clone propagated in vitro can express dual functions of cytokine secretion and cell killing.
Immediate cytotoxicity by circulating HIV-specific CD8+ T cells does not correlate with short-term IFN-γ secretion.
The validation of the assay to measure cytolytic activity and antigen-induced secretion of cytokines concurrently, in combination with the successful measurements of ex vivo cytolysis by CTL clones from an HIV-infected patient, allowed us to next address the relationship between immediate cytolytic activity and secretion of IFN-γ by circulating HIV-specific CD8+ T cells. We selected 4 HIV-infected patients, 2 with spontaneous control of infection and 2 with chronic viremic disease, who each exhibited robust HIV-specific responses for the KK10-epitope (range [0.85%, 7.11%]) in their bulk CD8+ T cell populations, as measured by tetramer staining (Figure A). We isolated CD8+ T cells by negative selection from frozen samples of PBMCs from each patient, and coincubated them in arrays of microwells with fluorescently labeled, HLA-matched, KK10 peptide–pulsed B cell targets. Immediate cytotoxicity was scored by imaging the arrays after 6 hours, and the secretion of cytokines (IFN-γ, IL-2, and TNF-α) was determined by microengraving during the final 2 hours of the assay. The data for cytotoxicity and cytokine release were then correlated for each well and segregated based on the number of targets per well (1 or 2–6). The number of effector cells was allowed to range from 1 to 5 because the probability of multiple antigen-specific T cells being confined in a single well is low, and the mean occupancy of T cells per well was between 1 and 2 for each sample. (When antigen-specific cells constitute 5% of the population, statistically 9.5% of wells with two CD8+ T cells will contain a single antigen-specific cell, whereas both cells will be antigen-specific in only 0.25% of the wells. Thus, any observed function in a well with two effector cells is 38 times more likely to result from a single antigen-specific cell than from two.) After filtering the matched data to remove wells with targets that were dead at the initial time point (t = 0 hours), approximately 2,000–3,000 independent events per sample were identified containing effectors coincubated with single targets (E/T = 1:1–5:1).
Direct evidence that immediate cytotoxicity and short-term IFN-γ secretion are not correlated functions for ex vivo HIV-specific CD8+ T cells.
Finally, we also incubated the same B cell lines with ex vivo CD8+ T cells from all 4 patients, but without exogenous or irrelevant peptides, in order to verify that endogenous peptides presented by the EBV-transformed B cell lines did not induce confounding CD8+ T cell responses. In these experiments, the numbers of cytolytic events observed were indistinguishable from the number of spontaneous deaths of target cells from wells on the same arrays containing no T cells (P ≥ 0.90). These arrays also yielded fewer than 1 IFN-γ+ event per 5,000 wells (<0.02%) by microengraving. These data confirmed that processing and presentation of endogenous peptides by the target cells did not significantly affect the detection and quantification of HIV-specific CD8+ T cell responses for these subjects.
The frequency of HIV-specific IFN-γ+ CD8+ T cells from each subject determined by microengraving ranged from 0.6% to 0.9%. This result was comparable to the frequency of IFN-γ+ cells as measured by standard ELISpot upon stimulation with KK10 peptide ([0.12%, 0.38%]) (Supplemental Figure 4). Since both our assay and ELISpot measured frequencies of IFN-γ+ events that were smaller than the fraction of tetramer-positive T cells determined by flow cytometry, we also assessed the KK10-specific intracellular production of IFN-γ in PBMCs from all 4 patients by flow cytometry (Supplemental Figure 5). These data showed that a number of cells produced IFN-γ in an antigen-dependent manner, but not all of them labeled with tetramers, and that the total number of tetramer-positive events following antigenic exposure was less than that in the absence of peptide. These results further suggest that antigen-dependent functional responses do not necessarily correspond with tetramer labeling.
The number of effector cells mediating cytotoxicity in our assay ranged between 0.5% and 1.9% of CD8+ T cells and was comparable among all 4 subjects. Correlation of IFN-γ secretion and cytolytic activity for individual effector cells, however, indicated that reactive, antigen-specific CD8+ T cells encountering a single APC either released IFN-γ or lysed their targets (single function, [0.8, 2.0%]), but rarely mediated both functions (dual function, [0, 0.04%]) (Figure B). We applied 4 different statistical analyses to test the hypothesis resulting from our data that cytolytic activity and IFN-γ secretion occur discordantly. First, a randomized resampling analysis was used to estimate the correlation coefficient for IFN-γ secretion and cytotoxicity. All functional events detected in the wells provided a common pool from which a subset of events was drawn 50,000 times; the number of events in each subset was based on the expected number of antigen-specific events relative to spontaneous death or nonspecific cytokine release on the array. This test showed a strong anti-correlation between cytolysis and secretion (r < –0.95 for each subject) (Figure B). Second, we applied the hypergeometric distribution to test the significance of overlap in cytolytic activity and IFN-γ secretion. Using the number of dual function events observed from each sample (ndual), we calculated the probability that at least ndual events would be observed due to random overlap between cytolytic activity and secretion. For all samples, this probability was greater than 0.05, suggesting that none of the samples had significant overlap in cytolytic and IFN-γ secretion functionality. Third, we calculated the probability (P) that the rare events with dual functions resulted from the random overlap of the secretion of IFN-γ and spontaneous death of the target cell. For each subject, we found P > 0.3, further indicating that there is not a significant overlap between cytolytic and secretion behavior in individual effector cells. Finally, we analyzed wells with 1–5 effectors and more than one target. These data confirmed that few CD8+ T cells exhibited both functional activities concurrently ([0.17, 0.37%]) (Supplemental Figure 6). The number of IFN-γ+ cells that exhibited cytolytic activity did not increase significantly for any of the subjects with T > 1 (P > 0.4).
The magnitude and breadth of functional CD8+
T cell responses have been shown to depend on the concentration of antigen to which the cells are exposed (28
). We therefore performed a peptide titration experiment by integrated single-cell analysis using CD8+
T cells isolated from one of the elite controllers (Fw056) coincubated with B cell targets that were pulsed with a range of concentrations of KK10 peptide. For peptide concentrations greater than or equal to 500 ng/ml, the frequencies of cytolytic events were significantly greater than in the control (P
< 0.01; Fisher’s exact test, or, for large numbers of events, χ2
with Yate’s correction) and increased with concentration (Supplemental Figure 7A). All 3 non-zero concentrations of peptide (50 ng/ml, 500 ng/ml, 5 μg/ml) induced a consistent percentage of cells secreting IFN-γ ([0.49, 0.64%]), with the relative rates of secretion significantly higher for concentrations of 500 ng/ml and 5 μg/ml relative to that at 50 ng/ml (Supplemental Figure 7B). No such secretion events were scored in the absence of peptide, confirming a low rate of false-positive responses for secretion of IFN-γ with only endogenous peptide. The overlap of the cytokine secretion with cytolytic activity remained minimal.
To further investigate the relationship between HIV-specific cytotoxicity and IFN-γ secretion, we next conducted the combined assay for 2 patients with an alternate, robust CD8+ T cell response to A*02-SL9. Similar to the KK10-specific response, we found that SL9-specific CD8+ T cells showed moderate IFN-γ responses (0.4% and 0.5%). The frequency of CD8+ T cells capable of both cytolysis and IFN-γ secretion was again low (0.06% and 0.07%, Supplemental Figure 8).
The above studies indicate the ability to assess the functions of effector cells when the targeted epitope is known. To determine whether this assay might be useful when the cognate epitope is not known, we loaded the array of microwells with B cell targets (EBV transformed; derived from an HIV-infected subject) that had been pulsed with a pool of overlapping peptides spanning the entire Gag protein. We then added autologous CD8+ T cells and examined the antigen-specific dual functionality of these cells. Discordant responses were again observed, without requiring a priori knowledge of the epitopes targeted or the restricting HLA alleles. In wells with 1–5 effectors and a single target, 0.29% of the CD8+ T cells secreted IFN-γ and 0.95% lysed their target, but no effector cells were observed to both secrete IFN-γ and lyse a target (Supplemental Figure 9). Together, these data provide direct evidence that cytolysis can occur independently of secretion of IFN-γ and that this segregation of functional responses is not restricted to a particular epitope.