Biology subjects

Nayak, I.

Publications and source records attributed to Nayak, I..

6 recordsLinked to original sources

bioRxiv2025

Natural Killer Cell Receptor Signaling and Activation Depend on Cell Cycle Stages

Receptor signaling in Natural Killer (NK) cells leads to post-translational modification (e.g., phosphorylation) of sub-cellular signaling proteins within minutes of receptor stimulation that eventually give rise to diverse effector functions including cell proliferation. Recent single-cell mass cytometry (i.e., CyTOF) experiments in macrophages showed variations of abundances of phosphorylated signaling proteins across cell cycle states indicating a dependence of cell signaling kinetics on an order of magnitude slower kinetics (~ several hours) of cell cycle transitions. We investigated cell cycle dependence of NKG2D signaling kinetics in NK cells by CyTOF measurements performed on IL-2-treated NKG2D-stimulated primary human CD56dim NK cells. The CyTOF experiments revealed monotonic or semi-monotonic increases of the average protein abundances of the majority of signaling proteins such as pCrkL, pPLC{gamma}2, and pErk, and the degranulation marker protein CD107a with progressing cell cycle states at specific time points post-NKG2D stimulation; however, several proteins such as pVav1, pS6, and pAkt, and early activation marker protein CD69 also showed non-monotonic variations in the average abundances with progressing cell cycle states. We used minimal mathematical and computational models coupling signaling and cell cycle processes to show that non-monotonic variations in the signaling protein abundances with progressing cell cycle stages are likely to arise in situations where protein synthesis and degradation and signaling kinetics are actively regulated by cell cycle processes.

immunology↗

bioRxiv2025

A framework integrating multiscale in-silico modeling and experimental data predicts CD33 CAR-NK cytotoxicity across target cell types

Uncovering mechanisms and predicting tumor cell responses to CAR-NK cytotoxicity is essential for improving therapeutic efficacy. Currently, the complexity of these effector-target interactions and the donor-to-donor variations in NK cell receptor (NKR) repertoire require functional assays to be performed experimentally for each manufactured CAR-NK cell product and target combination. Here, we developed a computational mechanistic multiscale model which considers heterogenous expression of CARs, NKRs, adhesion receptors and their cognate ligands, signal transduction, and NK cell-target cell population kinetics. The model trained with quantitative flow cytometry and in vitro cytotoxicity data accurately predicts the short- and long- term cytotoxicity of CD33CAR-NK cells against leukemia cell lines across multiple CAR designs. Furthermore, using Pareto optimization we explored the effect of CAR proportion and NK cell signaling on the differential cytotoxicity of CD33CAR-NK cells to cancer and healthy cells. This model can be extended to predict CAR-NK cytotoxicity across many antigens and tumor targets.

cancer biology↗

bioRxiv2024

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy

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 Ca2+ 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 Ca2+ 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.

immunology↗

bioRxiv2024

Impact of IL-21 on Natural Killer cell proliferation and Function - a mathematical and functional assessment

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-{kappa}B 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-{kappa}B 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-{gamma} secretion against hepatocellular carcinoma cell lines. Our work provides a mathematical framework for interrogating NK cell activation for cancer immunotherapy.

immunology↗

bioRxiv2022

Quantification of differential toxin expressions and their relation to distinct lifespans of bacterial subpopulations associated with diverse host immune mechanisms

An assortment of robust intracellular defence mechanisms are critical for restricting proliferation of pathogens and maintaining sanctity of the cytosol. Defect in these mechanisms could be exploited by the pathogens for creation of a safe sanctuary which can act as a transient reservoir for periodic dissemination into the host. While residing inside the host cell, pore forming toxins secreted by the pathogens compromises the integrity of the vacuole and exposes the microbe to diverse intracellular defence mechanisms. However, the correlation between toxin expression levels and consequent pore dynamics, fostering pathogens intracellular life, remains largely unexplored. In this study, using Streptococcus pneumoniae (SPN) and its secreted pore forming toxin pneumolysin (Ply), as model systems, we explored various facets of host-pathogen interactions in host cytosol, governed by the toxin expression and the resultant pore formation. The extent of damage on the endosomal membrane was found to dictate subsequent interaction with different host endosomal damage sensors. This in turn governed the routes of SPN clearance, revealing multiple layers of defence mechanisms at hosts disposal for counteracting invaded pathogens. A subset of SPN population producing extremely low amount of Ply inflicted minimal damage to the endomembrane, precluding decoration by endomembrane damage sensors and significantly prolonging its intracellular persistence. Such long persisting bacterial population could be key for pathogenic transmission or ensuing invasive disease. Using time-lapse fluorescence imaging, we monitored lifespans of different pneumococcal population subsets inside host cells. After quantitative analysis of various timescales such as pore formation time, vacuolar or cytosolic residence time and total degradation time, we developed a mathematical model that could correlate these to intravacuolar accumulation of Ply monomers. By proposing events like pore formation and vacuolar degradation of SPN as first passage processes, our theoretical modelling yields estimates of Ply production rate, burst size, and threshold Ply quantities which triggers these outcomes. Collectively, we present a general method by which intracellular lifespans of pathogens could be correlated to differential levels of toxins that they produce.

microbiology↗

bioRxiv2019

Kinetochore capture by spindle microtubules: why fission yeast may prefer pivoting to search-and-capture

The mechanism by which microtubules find kinetochores during spindle formation is a key question in cell biology. Previous experimental studies have shown that although search-and-capture of kinetochores by dynamic microtubules is a dominant mechanism in many organisms, several other capture mechanisms are also possible. One such mechanism reported in Schizosaccharomyces pombe shows that microtubules can exhibit a prolonged pause between growth and shrinkage. During the pause, the microtubules pivoted at the spindle pole body search for the kinetochores by performing an angular diffusion. Is the latter mechanism purely accidental, or could there be any physical advantage underlying its selection? To compare the efficiency of these two mechanisms, we numerically study distinct models and compute the timescales of kinetochore capture as a function of microtubule number N. We find that the capture timescales have non-trivial dependences on microtubule number, and one mechanism may be preferred over the other depending on this number. While for small N (as in fission yeast), the typical capture times due to rotational diffusion are lesser than those for search-and-capture, the situation is reversed beyond a certain N. The capture times for rotational diffusion tend to saturate due to geometrical constraints, while those for search-and-capture reduce monotonically with increasing N making it physically more efficient. The results provide a rationale for the common occurrence of classic search-and-capture process in many eukaryotes which have few hundreds of dynamic microtubules, as well as justify exceptions in cells with fewer microtubules.

biophysics↗