ABSTRACT
NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2 + influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca + + influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2 + flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70. Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.
ABSTRACT
Natural killer (NK) cells are currently in use as immunotherapeutic agents for cancer. Many different cytokines are used to generate NK cells including IL-2, IL-12, IL-15 and IL-18 in solution and membrane bound IL-21. These cytokines drive NK cell activation through the integration of STAT and NF-κB pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy between STAT3 and NF-κB transcription factors. Use of IL-21 in solution in the priming, but not post-priming phase of NK cell culture resulted in optimal NK cell proliferation, without compromising cytotoxicity or IFN-γ secretion against hepatocellular carcinoma cell lines. Our work provides a mathematical framework for interrogating NK cell activation for cancer immunotherapy.
ABSTRACT
Following invasion of the host cell, pore-forming toxins secreted by pathogens compromise vacuole integrity and expose the microbe to diverse intracellular defence mechanisms. However, the quantitative correlation between toxin expression levels and consequent pore dynamics, fostering the intracellular life of pathogens, remains largely unexplored. In this study, using Streptococcus pneumoniae and its secreted pore-forming toxin pneumolysin (Ply) as a model system, we explored various facets of host-pathogen interactions in the host cytosol. Using time-lapse fluorescence imaging, we monitored pore formation dynamics and lifespans of different pneumococcal subpopulations inside host cells. Based on experimental histograms of various event timescales such as pore formation time, vacuolar death or cytosolic escape time and total degradation time, we developed a mathematical model based on first-passage processes that could correlate the event timescales to intravacuolar toxin accumulation. This allowed us to estimate Ply production rate, burst size and threshold Ply quantities that trigger these outcomes. Collectively, we present a general method that illustrates a correlation between toxin expression levels and pore dynamics, dictating intracellular lifespans of pathogens.
Subject(s)
Longevity , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolism , Streptolysins/metabolism , Cytosol/metabolism , Bacterial Proteins/metabolism , Biological Transport , Host-Pathogen InteractionsABSTRACT
The first passage search of a diffusing target (prey) by multiple searchers (predators) in confinement is an important problem in the stochastic process literature. While the analogous problem in open space has been studied in some detail, a systematic study in confined space is still lacking. In this paper, we study the first passage times for this problem in one, two, and three dimensions. Due to confinement, the survival probability of the target takes a form â¼e^{-t/τ} at large times t. The characteristic capture timescale τ associated with the rare capture events are rather challenging to measure. We use a computational algorithm that allows us to estimate τ with high accuracy. We study in detail the behavior of τ as a function of the system parameters, namely, the number of searchers N, the relative diffusivity r of the target with respect to the searcher, and the system size. We find that τ deviates from the â¼1/N scaling seen in the case of a static target, and this deviation varies continuously with r and the spatial dimensions.
ABSTRACT
First passage in a stochastic process may be influenced by the presence of an external confining potential, as well as "stochastic resetting" in which the process is repeatedly reset back to its initial position. Here, we study the interplay between these two strategies, for a diffusing particle in a one-dimensional trapping potential V(x), being randomly reset at a constant rate r. Stochastic resetting has been of great interest as it is known to provide an "optimal rate" (r_{*}) at which the mean first passage time is a minimum. On the other hand, an attractive potential also assists in the first capture process. Interestingly, we find that for a sufficiently strong external potential, the advantageous optimal resetting rate vanishes (i.e., r_{*}â0). We derive a condition for this optimal resetting rate vanishing transition, which is continuous. We study this problem for various functional forms of V(x), some analytically, and the rest numerically. We find that the optimal rate r_{*} vanishes with a deviation from the critical strength of the potential as a power law with an exponent ß which appears to be universal.