Natural Killers at the Neuro-Immune Axis!

Much has been said about the role Natural Killer (NK) cells play in positively and negatively influencing events in tissues and cells. Summarized facts about the healthy state of NK cells in humans and animals explain how innate immune cells, including NK cells differ from adaptive immune cells. One significant feature of NK cells is that they can act independently of MHC antigen presentations and that makes them tantalizing, but enormously challenging for scientists seeking to embrace their  influence over cells and their killing capabilities.

The variety and combination of inhibitory and activating receptors differentially expressed by as many as 30,000 human NK cell subsets makes heterogeneity difficult to relate across different conditions, organs and tissue types. Notwithstanding, positive rates of overall patient survival resoundingly corelated to the presence of as few as one NK cell infiltrating a tumor in a microscopic field.  

Innate immune cells including NK and macrophages have also been directly tied to conditions of neurological pain and more specifically to afferent and efferent fibers that signal through the vagus nerve. In these models at the immune-neurological interface similarities exists and both organs must interact for proper function. 

In each of these organs communication is mediated by direct cellular contact eg. synapse formation and via soluble mediators like cytokines or neurotransmitters that also communicate bi-directionally between cells of each system. The nervous and immune systems can influence each other’s activity because immune cells express neurotransmitter receptors, and neurons express cytokine receptors. Immune cells can synthesize and release neurotransmitters themselves, thus using neurotransmitter-mediated pathways via autocrine and paracrine mechanisms. This may indicate that NK cells extend nerve end signaling further into tissues and at a cellular level. 

A recent paradigm in physiology describes the existence of neuro-immune cell units, at an organ-tissue level and identifies the enormous complexity inherent in this globally unifying approach that also connects neuro-immune-gut, at least in Parkinson's disease.

Parkinson’s is a brain disorder where certain nerve cells slowly die and symptoms worsen. The risk of developing the condition increases with age, but in certain patients the illness is caused by defects in two proteins, PINK1 and parkin. NK cells are capable of homing to the central nervous system in neurological disorders that exhibit exacerbated inflammation and inhibit hyperactivated microglia. Recently, a study demonstrated that NK cells scavenge alpha-synuclein aggregates and systemic depletion of NK cells results in exacerbated neuropathology in a mouse model of alpha-synucleinopathy, making NK cells highly relevant in Parkinson’s disease.

We recently described a mechanism by which the sentinel state of NK cells is impaired and suggested the senescent phenotype, induced by age related mitophagy could be the primary cause. Increase in mitophagy (mitochondrial autophagy) is age-dependent and abrogated by PINK1 or parkin deficiency suggesting, in Parkinson's disease compromised mitophagy is associated with neurological degeneration. Further  PINK1 and Parkin, which are regulated by p53 specifically repress mitochondrial antigen presentation of both MHC classes. Therefore, excessive PINK1 or parkin increases rates of NK cell mitophagy and repress the presentation of mitochondrial antigens for MHC classes at the axis of this neuro-immune related disease.

The healthy state of NK cells at the axis of neuro-immune systems may indeed have more far reaching implications for the future of human diseases and therapies.

 

Natural Killers Linked to Overall Survival in Cancer

A meta analysis of tumor samples, collected between 1973 and 2016, in 53 studies confirmed overall survival (OS) correlated with Natural Killer cell infiltration into solid tumors. The number of NK cells infiltrating solid tumors, including those considered “highly ”infiltrated was relatively low, compared with other immune populations. Notwithstanding, the presence of a single NK cell, within a high powered microscopic field was associated with significantly improved OS and disease free survival in colorectal cancer, HER2 + breast cancer and hepatocellular carcinoma.

The finding supports the prospect that single tumor infiltrating NK cells, in a sampled tissue can be determinative for OS. By inference a single tumor infiltrating NK cell or cells possess characteristics that are relative to OS and beneficial to patient.  

NK cell surface receptors are densely varied defining at least 30,000 unique NK cell populations within each individual. NK cell classifications, relative to tumor infiltration and OS is enormously complex, especially at this scale and present definitions of activating and inhibiting receptor combinations underwhelm. To identify NK cells that have infiltrated or may be capable of infiltrating a patient tumor to improve OS we focused on biopsied tumor tissue selections whether or not they include NK cells.

Our work is with two tumor types in humanized mice. Multiple sections of each tumor were resected and divided into multiple parts for coculture with allogenic naïve, IL2 and probiotic enhanced NK cells and for DNA sequencing. After coculture NK cell cytotoxicity and other detailed measures resulting from each resected section and from single cells were assessed. Presently sequencing of DNA from each resected, divided section (pre-coculture) is focused on comparisons derived from TP53.

In the final stage NK cells will be cocultured with resected tumor tissue and will be made to challenge new tumor tissue and single cells, from the resected tumor from which the NK coculture was derived. The objective will be whether Codondex analysis of TP53 DNA sequencing can predict the most successful tumor tissue candidates based upon the most effective cocultured NK cell challenge to the tumor derived tissue or cells. 

If Codondex algorithm is found to identify a direct or indirect logic for tissue or cell selection that is effective in vitro our work will continue to next stage in vivo testing and analysis on similar grounds. 

  

 

Not Only A Killer A System for Killing!

The next time you're out exercising, spare a thought for your busy mitochondria. NASA scientists just reported mitochondria as the key to health problems in space.

Natural killer (NK) cells can extend membrane probes into cells or pathogens. These are loaded with granulysin (GNLY) to penetrate and perforin (PFN) to kill intracellular bacteria or protozoa and can lyse entire cells. The probes can also transfer healthy mitochondria to apoptotic cardiomyocytes (and other cells) in need of mitochondrial transfer. Uterine NK cells of the decidua send probes into trophoblasts to selectively kill intracellular Listeria monocytogenes without killing the trophoblast host. Stressed cells, moving toward apoptosis can behave similarly, but in reverse shooting out nanoprobes to proximal cells seeking cooperation and urgent mitochondrial transfers including to cancer cells.

A meta-analysis of gene expression signatures for blood pressure and hypertension in 7017 individuals from 6 international studies found of 7717 genes, 34 were most differentialy expressed including GNLY. Enrichment analysis for the diastolic and systolic gene group's associated strongly with NK cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response.

Formation of membrane probes or tubes, in which mitochondria travel and establishment of intracellular mitochondrial networks in the peripheral zone of cells require Kinesin-1 heavy chain (KIF5B). KIF5B is also required for female meiosis (oogenesis) and proper chromosomal segregation in mitotic cells and modulates central spindle organization in late-stage cytokinesis in chondrocytes.

A study of centromere heterochromatin (connected with central spindle) surprisingly showed that distant euchromatic regions, enriched in repressed methylated genes also interacted with the hierarchical organization of centromeric DNA. These 3D spatial interactions (at a distance) are likely mediated by liquid-like fusion events and can influence the health of individuals. Repressed gene's were identified as transposable elements, sequences often associated with pathogenic DNA insertions that have been persistently retained.  

KIF5B is an interaction partner of ADP-ribosylation factor-like 8b (Arl8b), which is required for NK cell–mediated cytotoxicity that drives polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between NK and target cells. Silencing experiments that led to failure of MTOC-lytic granule polarization suggest Arl8b and KIF5B together control the critical step in NK cell cytotoxicity. 

KIF5B is also a critical transporter of p53 and c-Myc to the cytoplasm for degradation. However, subcellular localization of Arl8b and p53-dependent cell death was shown to occur through knockdown of acetylation subunit NatC. As a consequence, p53 is stabilized, phosphorylated and significantly activates transcription of downstream proapoptotic genes. In the absence KIF5B, or presence of  mutants p53 and c-Myc aggregate in the nucleus where they signal DNA damage-induced apoptosis through the control of p53 by endogenous c-Myc (in vivo).

Finely tuned, frequently used KIF5B in NK cells expressing GNLY may induce effects on local tissue blood pressure, as was discovered by expression of Renin-Angiotensin vasoactive proteins AT1, AT2, and ANP in pregnancy-induced uterine NK cells. Inflammation signaling, via tissue bound NK cells may result from stretch-mediated release of angiotensin II, which is coupled with p53 acetylation apoptosis and activation of p53. This may prolong upregulation of the local renin-angiotensin system, increase susceptibility of target cells to apoptosis and signal adaptive immune cells. 

Somewhere in the balance between NatC knockdown induced apoptosis and angiotensin II induced apoptosis p53 may direct traffic to keep your cells healthy!

 

An Integrated P53 Puzzle - Glycolysis in Cancer, Diabetes and Immunity!

Oxygen poor, hypoxic tissue promotes a cellular shift in mitochondrial metabolism from OXPHOS to less energy efficient glycolysis. Each shift induces environmental, epigenetic and genetic factors that alter a cells response to insult, attack and disease. Endothelial tip cells at micro-vessel ends are predominantly glycolytic. However, deletion of PFKFB3, the critical regulator of glycolysis reduced the sprouting of micro-vessel tips and elevated PFKFB3 levels improved tip cell sprouting, direction and cell behavior.

In response to DNA damage p53 promotes nucleotide biosynthesis by repressing the expression of PFKFB3. This increases the flux of glucose, through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient repair of DNA damage and cell survival.

In Panc1 pancreatic cells, pro-apoptotic TGFβ1 enhanced PFKFB3 expression and stimulated glycolysis. Extracellular lactate induces endothelial mesenchymal transition (EMT) by remodeling the extracellular matrix and releasing activated TGFβ1.  TGFβ is a potent immunosuppressive cytokine that can impede development and function of natural killer (NK) and other immune cells. Furthermore, high extracellular lactate levels can contribute to immune evasion, thereby promoting tumor growth and metastasis. In tumor microenvironments glycolysis also leads to accumulated lactate, which stabilizes hypoxia inducible factor 1α (HIF-1) and upregulates the expression of anti-apoptotic, VEGF (in axis with NRP-1 dependency) resulting in angiogenesis and stimulation of cell migration. 

Hypoxia induces the loss of differentiation markers of several tumor types while increasing expression of embryonic markers such as transcription factors NANOG, OCT4, SOX2, and the Notch ligand. This reprogramming, toward a cancer stem phenotype is associated with increased tumorigenesis. In non-small cell lung carcinoma cells hypoxia increased NANOG expression that contributed to hypoxia-induced tumor cell resistance against cytotoxic lymphocyte (CTL)-mediated lysis.

Under stress the outer mitochondrial membrane incorporates Pink1, which binds and phosphorylates p53 at serine 392 and aids phagophore formation to enhance mitophagy. This reduces transport of p53-s392 to the nucleus where it would otherwise disrupt transcription of Nanog. p53 regulates Pink1 and Parkin, which regulate mitochondrial antigen presentation of both MHC classes. 

The development of type 1 diabetes involves a complex interaction between pancreatic β-cells and cells of the innate and adaptive immune systems. Analyses of the interactions between NK cells, NKT cells, dendritic cell populations and T cells have highlighted how these can influence the onset of autoimmunity. NK cells were observed in the pancreas, in NoD mice before T cell infiltration and are critically required in the pancreas for accelerated diabetes.

The islet in type 2 diabetes (T2D) is characterized by IAPP amyloid deposits, a protein co-expressed with insulin by β-cells. Human IAPP (hIAPP) misfolded protein stress activates HIF-1/PFKFB3 signaling, which increases glycolysis, mitochondrial fragmentation and perinuclear clustering, considered protective against increased cytosolic Ca2+, characteristic of amylin toxic oligomer stress. β-cells in adult humans are minimally replicative and fail to execute the second pro-regenerative phase of the HIF-1/PFKFB3 injury pathway. β-cells remain trapped in the pro-survival first phase of the HIF-1 injury repair response with a metabolism and mitochondrial network adapted to slow the rate of cell attrition at the expense of β-cell function. The senescent-like state may support the reduced NK cell activity and presence of more pro-inflammatory M1 macrophages in T2D

p53 deficient tumors can be metabolically reprogrammed and regressed by deleting isoforms of p63 or p73 to upregulate IAPP and amylin, which through the calcitonin receptor (CalcR) and receptor-activity-modifying-protein 3 (RAMP3) inhibit glycolysis, induce ROS and apoptosis. In epidermal keratinocytes p63 promotes glycolytic metabolism  by binding PFKFB3 consensus sites required for mRNA and protein expression.

Senescent cells typically upregulate anti-apoptotic pathways, and are preferentially susceptible to inhibition of these pro-survival mechanisms. This has been dubbed the ‘Achilles heel’ of senescent cells and may relate to the low mitochondrial membrane potential found in many senescent cells that ease the release of apoptosis-stimulating factors from mitochondria to promote survival. Similar weaknesses may be present through glycolysis in cancer, diabetes, other diseases and immune response.

p53 in Transition, Covid19, Cancer and Immunity

p53's trajectory, sensitivity and function influences different outcomes in stages of transition of developing pluripotent or embryonic stem cells that can inform tumorigenesis and immune response. 

Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation, and DNA damage-induced apoptosis was p53-independent. 

Human (induced) pluripotent stem cell differentiation, from endoderm toward mesoderm was driven by a DNA damage-induced, time-sensitive, p53 transcriptional program. In cells passing through epithelial-to-mesenchymal transition DNA damage prevents the normal reduction of p53 levels, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. 

From the blastocyst, villous cytotrophoblasts undergo a partial epithelial to mesenchymal transition (EMT) when they differentiate into extravillous cytotrophoblasts and gain the capacity to migrate and invade. Extravillous cytotrophoblast invasion involves a cellular transition from an epithelial to mesenchymal phenotype. TWIST, an emerging gene of interest strongly influences p53 to complete EMT.  

p53 is necessary for cells to initiate EMT, but attenuation of its levels by MDM2 is also necessary for expression of the mesenchymal phenotype. Downregulation of p53 may be directly controlled by this transition as the EMT factor TWIST1 can bind p53 leading to its MDM2-dependent degradation. During definitive endoderm differentiation, downregulation of p53 may be necessary for the normal transcriptional program to proceed. The unscheduled stabilization of p53, caused by DNA damage may result in a transcriptional perturbation driving differentiation away from definitive endoderm.

Using KRAS-driven pancreas tumor-derived cancer cells as a model of p53 loss, p53 deletion can promote immune tolerance through the recruitment of both myeloid and Treg cells. Enrichment of these suppressive cell populations enhanced the protection of p53-null cancer cells from immune-mediated elimination. 

Tumor-derived VEGF through VEGFR2 and NRP-1 creates a perivascular niche to regulate the initiation and stemness of skin tumors and autocrine VEGF promotes survival and invasion of prostatic, pancreatic cancer and glioblastoma cells, particularly for cancer stem-like cells in a NRP-1-dpendent enhanced EMT manner. 

A recent SARS-CoV2 update may point to anti-apoptotic affects that occur through the axis inactivation of p53 and mitochondrial apoptotic pathway as mediated by NRP-1, in endothelial cells of Zebra Fish. Decreased levels of p53 might suppress caspase cleavage and therefore downregulate apoptosis (a feature of Covid19). Data showed that p53 is the downstream signaling molecule of PI3K/Akt pointing at MDM2 as a signaling component in NRP-1 survival signaling. NRP-1 was shown as a host factor for SARS-CoV-2 infection and in a successful Covid19 phase trial, for critical care patients injection of apoptotic cells induced signaling to restore immune homeostasis.  

Even brief reactivation of endogenous p53, in p53-deficient tumors can produce complete tumor regressions. Primary response to p53 reactivation was not apoptosis, but the induction of a cellular senescence program associated with differentiation and upregulation of inflammatory cytokines. 

Elimination of senescent tumors, by Natural Killer (NK) cells occurred as a result of signal cooperation associated with p53 expression or senescence, which regulate NK cell recruitment and other signals that induce NKG2D ligand expression on tumor cells. p53 expression enhances CCL2-dependent NK cell recruitment to the tumors.

A feature of several NK cell activating receptors resides in their capacity to detect self molecules induced in conditions of cellular stress. This is the case for NKG2D, which interacts with various ligands, including CCL2 that are expressed at low levels in most tissues but are overexpressed upon initiation of cellular distress, for example, after initiation of the DNA damage response.

Codondex is working to identify p53 status in cells isolated from TME tissue samples that can be cocultured to educate NK cells to stimulate a desired immune response. 

p53 vasoregulation and NK cell depletion in SARS-CoV2

p53 has earned first prize in the academic stakes. It is also the most mutated gene in cancer and elephant's have 20 copies, which probably explains their surprisingly low rate of cancer. Its associations to innate immunity, particularly Natural Killer (NK) cells through the mechanics of vasoconstriction-dilation have become a point of interest in COVID19 patients.

Remarkably COVID19 has inspired the global scientific community to focus a significant portion of its aggregate research toward the impact of  SARS-CoV2 (CoV2). For the first time in history global research is singularly focused because a large number of other protein's and gene's are affected by CoV2 binding Ace2. The Ace2 receptor is important in systems of vasoconstriction-dilation and has wide ranging impact.

CoV2 binding Ace2 reduces its availability to convert Angiotensin1 to Angiotensin 1-7 (Ang1-7) or Angiotensin 1-9 (Ang1-9), which primarily interact via MAS and Angiotensin2 Receptor (AT2R) respectively. These have been linked to signaling and stretch caused by vasoconstriction-dilation, mitochondrial dysfunction, mitochondrial fission as well as cardiac and vascular remodeling.

Ang1-7 and Ang1-9 interactions with MAS or AT2R cell surface receptors have been linked to signaling events that drive p53 binding DNA and transcription. Myocyte stretching activates p53 and p53-dependent genes, leading to the formation of Angiotensin II (Ang II) and apoptosis. AngII, stimulates phosphorylation of p53 (on serine 15) and CREB (on serine 133) and signaling converges on the p53-CRE enhancer to stimulate Bradykinin receptor 2 (BK2) gene transcription. BK2 is a key element in the p53 related kallikrein-kinin system (KKS) of vasodilation that counters the Renin-Angiotensin-Aldosterone-System (RAAS) of vasoconstriction. 

Aldosterone was shown to induce mitochondrial dysfunction and podocyte injury mediated by p53/Drp1-dependent mitochondrial fission. In neuronal cells p53 dependent declines in Drp1 and parkin contribute to altered mitochondrial morphology and cell death. Parkin, via Pink1 activity binds depolarized mitochondria to induce autophagy of mitochondria. Mutations in both Drp1 and Pink1 were fatal in Drosophila models. These events also implicate a direct functional link to chronic inflammation in ageing between p53 and expression levels of ICAM1 on endothelial and NK cells required to bind targets. The p53 mediated negative regulation of autophagy is Pink1 dependent and experiments have shown that mitochondrial antigens, recognized by NK cells presented on MHC's are Pink1 and parkin dependent. 

Severe COVID-19 patients have highly elevated Bradykinin and AngII, perhaps an indication of elevated p53 trends that have been discovered in these patients. Under normal circumstances, on endothelial cells Bradykinin would act as a potent vasodilator via its BK2 receptor. However, since Ang (1-7) potentiates Bradykinin action on BK2 receptors its near absence may reduce KKS vasodilation. On the other hand RAAS, also via p53 and elevated AngII primarily interacts with AT1R to promote vasoconstriction.  

NK cells through their Renin Angiotensin System may counter-regulate target cells in response. However, in COVID19 patients depletion of NK cells, invasion of Neutrophils and endothelial cell damage, in part through elevated p53 autophagy and apoptosis is the overwhelming nasty work of CoV2 against the backdrop of dysregulated blood pressure in tissue.

 

 

$100,000 Biohunt

Some of the past research on neoantigen and p53 antibodies in immunity has been encouraging. The data is enormously complex, but keeps pointing to TP53's great potential. To this end, we were anxious to start our mega-experiment, but were delayed by C19, now I'm glad to report we are well underway. In co-operation with researchers at UCLA we aim to determine whether Codondex transcript analysis, of TP53 can predict the best tumor tissue selection for most effective Natural Killer (NK) cell priming, activation and cell killing, including in autologous tumor micro environments.

We're hoping to to achieve a result along the path toward our ambitious clinical goal. We aim to prove that a specifically selected section from biopsied tissue can be used to effectively prime autologus NK cells for patient reapplication and disease treatment. 

This co-culture vs. sequencing challenge uses sections (T1-T8) taken from each of two tumors. Each section is co-cultured with 2 treated NK cell and one naive NK cell line and tests the efficacy of NK cell cytotoxicity against tumor cell and tumor tissue in killing assays. Separately, by sequencing TP53 of each selection and computing Codondex iScore(TM) algorithm we hope to identify specific features of each tissue selection that point computed results to research outcomes.

Co-culture vs. Sequencing Challenge

To better understand the analysis and encourage research contributions we are inviting applicants for first grants directed toward this objective. 

Codondex tools analyse genetic sequences at an arbitrary number of nucleotides. The tool provides an easy way to observe fine repetitive details of small subsequences contained within a gene. We compute various metrics for each subsequence including 'Inclusiveness', which measures the total occurrences of every computed smaller subsequence is found within the subsequence of interest. 

Our primary interest is intronic, non-coding DNA in multi-transcript genes. In these systems we create a transcript list, which we call the Vector, that is sorted by Codondex i-Score. This metric looks at Inclusiveness scaled by the length of the subsequence, to better account for intrinsic probability of finding smaller subsequences within progressively longer ones. Using this we look at the way order of this vector changes from subsequence to subsequence. Large changes in these vectors then prompts us to tag them for further investigation as it represents large deviation from transcript similarity, with this subsequence being labelled a Key Sequence. 

Codondex is proposing 3 grants for open problems to aid in our journey towards a more biologically useful platform. These 3 problems span statistical analysis, data acquisition and biological relevance of various aspects that are integral to our platform. 

Applicants should inquire further and sign up here.