Quantitative High-Throughput Single-Cell Cytotoxicity Assay for T cells

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 trials1-4. 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 toxicity5. 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 vivo6,7. 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 targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. 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

Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR

High-Throughput Single-Cell Functional and Molecular Profiling of Immune Cells in Cancer Immunotherapy

Immunotherapy has revolutionized the treatment of cancer and newer approaches including the adoptive transfer of genetically modified T cells reprogrammed to target tumor antigens have shown remarkable responses. Despite their promise, the efficacy of adoptive immunotherapy remains unpredictable due to the heterogeneous nature of the infusion products, patients’ characteristics, treatment regimens, and tumor burdens. Specifically with regards to the T-cell infusion product, there is a need to develop methodologies that allow for definition of potencies to understand the phenotypic, molecular, and functional contribution of infusion products at single-cell level. In the first part of this dissertation, we implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to demonstrate that while CD4+CAR+ (CAR4) cells killed at slower rate, most likely due to lower granzyme B content, they benefited from apoptosis resistance compared to CD8+CAR+ (CAR8) cells. These findings suggest that overall potency of multi-killing should be evaluated together in their context to resist apoptosis. In the second part of this dissertation, we developed single-cell multiplexed platforms comprising beads biosensors for detecting protein secretion, TIMING to monitor motility and cell-cell interactions, and microfluidic qPCR for transcriptional profiling. Analysis of thousands of single-cell interactions for over 5 hours revealed that the integrated behavior of polyfunctional T cells that kill and secrete IFN-γ was similar to those without IFN-γ secretion, suggesting cytolysis to be the dominant determinant of the interaction behavior and that killing enables faster synapse termination. In addition, tracking the speed of these cells by TIMING indicated that serial killer T cells may be identified based on their high out-of-contact basal motility. Transcriptional profiling of these single-cells confirmed that the motile cells expressed increased amounts of perforin and displayed an activated phenotype. In summary, these results highlight the heterogeneity of immune cells and thus, the need for definition of potency prior to infusion. We propose that single-cell platforms as demonstrated here are suitable to uncover the diversity and to help identify optimal functional and molecular biomarkers for applications in the clinic.Chemical and Biomolecular Engineering, Department o

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

Key Points • Fc-engineered mAb promotes NK cell ADCC via better activation, serial killing, and kinetic boosting at higher target cell densities. • Enhanced target killing also increased frequency of NK cell apoptosis, but this effect is donor-dependent. The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effectormediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cellmediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. (Blood. 2014;124(22):3241-3249

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells

<div><p>Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled <i>ex vivo</i> revealed that CD56^dimCD16⁺ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.</p></div

The frequency of IFN-γ-secreting T cells enumerated by functionalized beads within nanowell arrays is correlated to the same responses determined using ELISPOT.

<p>(A) Background-corrected mean fluorescence intensity (MFI) detected from a minimum of 30 IFN-γ-positive beads, as a function of IFN-γ analyte concentration with functionalized LumAvidin^® beads, determined on nanowell arrays. (B) Comparison of the bead assay against ELISPOT for detection of single effector cells (PBMC or TIL) secreting IFN-γ at varying level of antigenic stimulation (viral peptide pools or PMA/ionomycin). Linear regressions show that both approaches are significantly correlated (R² = 0.87, <i>p</i>-value = 0.0008).</p

NK cells that secrete IFN-γ early have higher CD16 surface expression.

<p>(AB): The distributions (black columns) of t_Secrete of single-NK cells were positively skewed, indicating the existence of a faster secretor subpopulation within the population of NK cells that secrete IFN-γ. The corresponding normal distributions (red curve) were plotted using the same mean and standard deviation of t_Secrete of single-NK cells. The relative comparison of CD16 (C) or CD56 (D) surface expression of early secretors (t_Secrete < population mean) and late secretors (t_Secrete > population mean). (E) The amount of IFN-γ secreted by NK cells during the 6 h period of observation was statistically different across two donors. The amount of IFN-γ secreted is inferred from the ratio of fluorescent intensities (ratio of maximum and minimum value) from the fitting curve; (F) The relative IFN-γ secretion rate was a donor-dependent parameter. The rate of secretion of IFN-γ was inferred from the Hill slope (MFI versus time) obtained from curve fit on two different donors (donor 1: light red; donor 2: dark red). Error bar: mean and 95% confidence intervals are shown. Mann-Whitney test was performed, ns: not significant, ***: <i>p</i>-value < 0.001, ****: <i>p</i>-value < 0.0001.</p