Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma

Abstract

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

Interaction kinetics with transcriptomic and secretory responses of CD19-CAR natural killer-cell therapy in CD20 resistant non-hodgkin lymphoma

“…CD19 antigen density were determined by flow cytometry, using Quantibrite anti-CD19-PE (Clone SJ25C1)#340364, and Quantibrite-PE calibration beads (BD Bioscience, San Jose, CA, USA). Primary human lymphoma cells (EL-2 and EL-5) were isolated from IRB exempt discarded tissue specimens of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) obtained through Tufts Tumor Repository, as described previously [17]. Activated NK-92 (aNK) and CD19.CAR.NK92 cells were provided as gift from Nantkwest, Inc. (Woburn, MA, USA) and the details of CAR construct used in design and development of CD19.CAR.NK92 cells is outlined in [1,2], and (Supplemental Methods).…”

“…Target cells labeled with 2μM Calcein AM (Thermo Fisher, Waltham, MA; excitation/ emission: 495nm/515nm) at 37°C for 20 minutes, were washed once with media and loaded in a syringe at 1.5 million/mL concentration. Both target cells and unlabeled CD19.CAR.NK92 were introduced in the microfluidic device, oil droplets were generated and image acquisition were performed as outlined before [17]. Target cell death was indicated by significant (>80%) loss of Calcein AM fluorescence, number of contacts made and duration of contacts between cell pairs were determined as described previously [17,22].…”

Integrating Immunology and Microfluidics for Single Immune Cell Analysis

“…Their results suggested that the activation of single T-cells is faster when cells come in contact with dendritic cells in comparison to other activation methods. Furthermore, they developed a method to probe into the potentially heterogeneous cytolytic behavior of human NK cells ( 162 ). They demonstrated a 100% killing efficiency of NK cells, which is in contrast to earlier findings by various groups performed either in bulk or single cell ( 105 , 163 ).…”

Evaluation of cytotoxic T lymphocyte-mediated anticancer response against tumor interstitium-simulating physical barriers

“…The Voldman's group has published a series of works regarding pairing cells using hydrodynamic traps, which allowed the deterministic control of the initiation of cell-cell interactions, including CTL and the antigen-presenting cells 50,51 . Sarkar et al probed the cytolytic activities of individual NK cells coencapsulated with lymphoma cells in picoliter droplets generated by a microfluidic device 52 and demonstrated that a higher efficiency of the derivative NK cell line NK-92 in eradication of primary B-cell non-Hodgkin lymphoma cells than the primary NK cells. Christakou et al examined the variation of one hundred NK-mediated cytolysis against major histocompatibility complex class (MHC) I-deficient tumor cells in parallel by utilizing ultrasound to synchronize the contacts between the two types of cells in a multiwell chip 53 .…”