Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Abstract

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials [1][2][3][4] . There are several advantages to using CAR + T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR + T cells in the event of host toxicity 5 .Delineating the optimal functions of CAR + T cells associated with clinical benefit is essential for designing the next generation of clinical trials.Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo 6,7 . While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets 8 .Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes 9 . In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells. Video LinkThe video component of this article can be found at http://www.jove.com/video/50058/ Protocol 1. Reagents Preparation 1. Prepare RPMI-PLGH by mixing 500 ml RPMI-1640 and 5 ml each of Penicillin-streptomycin, L-glutamine, and HEPES solution. 2. Prepare R10 solution by mixing RPMI-PLGH with 10% Fetal Bovine Serum (FBS). The FBS is heat-inactivated at 56 °C for 30 min prior to addition. 3. Pre-warm at 37 °C 50 ml of RPMI-PLGH, 15 ml of PBS, and 15 ml of R10 in sterile conical tubes. 4. Fabrication of arrays of nanowells in PDMS: The silicon master is fabricated using photolithography essentially as described previously 10 . Mix thoroughly the Sylgard 184 elastomer kit base and curing agent at 10:1 weight ratio in a disposable cup using a plastic knife. Degas the mixture in a vacuum chamber for 1 hr. Pour mixture onto the nanowell array, seal with a glass slide and let it sit for 30 min. Transfer the assembly into an oven set...

Multifaceted Role of BTLA in the Control of CD8+ T-cell Fate after Antigen Encounter

“…Target cell cytolysis mediated by TIL was monitored using a Carl Zeiss Axio Observer fitted with Hamamatsu EM-CCD camera using 10 × 0.3 NA objective. Apoptotic cells became green when stained with Annexin V conjugated with Alexa 647as previously described (22). …”

Defining potency of CAR+ T cells: Fast and furious or slow and steady

“…We developed a high throughput in vitro Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) platform which allows the interrogation of thousands of single-cell level interactions between T cells and tumor cells in confined nanowells. [6][7][8] Specifically, we compared the potency and kinetics of CD19-specific CD8 + T CAR + T cells (CAR8) and CD4 + CAR + T cells (CAR4) in their interactional behavior with CD19 + tumor cells. 7 At E:T of 1: 2-5, we found that both CAR8 and CAR4 cells can participate in multikilling through conjugation to multiple tumor cells, although CAR4 cells experience a significant delay in kinetics of killing the tumor cells ( Figure 1A).…”

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

“…27 Fluorescently labeled annexin V was used to detect early apoptosis, and time-lapse microscopy using annexin V and Sytox (DNA dye) costaining confirmed that monitoring annexin V staining was sufficient to track eventual cell death (supplemental Video 1). Within nanowells containing a single NK cell and 1 to 3 targets, after 6 hours, 25% 6 8% (mean 6 standard deviation [SD]; n 5 5 donors) of NK cells induced apoptosis of DLETargets ( Figure 1I).…”

“…27 Briefly, CD33 1 EL4 (targets) and NK (effectors) cells were labeled for 5 minutes with 1 mM red PKH26 and green PKH67 (Sigma-Aldrich), respectively. The targets were subsequently incubated with 1 mg/mL mAb at 4°C for 20 minutes and were washed to remove excess mAb.…”

“…In vitro dynamic imaging systems are particularly suited for studying the dynamics of cell-cell interactions in a defined environment but have been typically limited in throughput and in monitoring effector fate. [25][26][27][28] We engineered the Fc region of the anti-CD33 mAb HuM195 by introducing the triple mutation S293D/A330L/I322E (DLE) and developed Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) to analyze ADCC kinetics of thousands of individual NK cells incubated with mAb-coated target cells. We demonstrate that mAb Fc engineering promotes superior activation of NK cells and improves both amplitude and kinetics of NK cell-mediated ADCC.…”