IFN-γ Concentration, p53 and Immune Sensitivity

IFN-γ 

Dimorphic complexity between Human Leukocyte Antigen (HLA) and Killer Immune Receptor (KIR) haplotypes introduce significant challenges for personalized Natural Killer (NK) and immune cell therapy. In vitro models support a p53 requirement for upregulation of NK ligands and there is a strong association between the KIR B haplotype and p53 alteration in Basal Cell Carcinoma's (BCC) with a higher likelihood that KIR B carriers harbor abnormal p53. Data suggests that KIR encoded by B genes provides selective pressure for altered p53 in, at least BCC's. 

Breast cancer (BC) patients exhibit reduced NK-cytotoxicity in peripheral blood. To test whether certain KIR-HLA combinations impair NK-cytotoxicity that predispose to BC risk, KIR and HLA polymorphisms were analyzed in 162 women with BC and 278 controls. KIR-B genotypes increased significantly in BC. Certain activating KIR (aKIR) HLA ligand combinations were significantly increased in advanced-BC patients whose combinations also shared specific inhibitory KIR (iKIR) counterparts. Contrarily, iKIR-HLA pairs without their aKIR-HLA counterparts were significantly higher in controls. The data suggests NK cells expressing iKIR to cognate HLA-ligands in the absence of specific aKIR counterpart are instrumental in antitumor response. 

The TP53 family consists of three sets of transcription factor genes, TP53, TP63 and TP73, each expresses multiple RNA variants and protein isoforms. TP53 is mutated in 25-30% of BC's, but the effect of isoforms in BC is unknown. Predicted changes in expression of a subset of RNAs involved in IFN-γ signaling were confirmed in vitro. Data showed that different members of the TP53 family can drive transcription of genes involved in IFN-γ signaling in different BC subgroups. Moreover, tumors with low IFN-γ signaling were associated with significantly poorer patient outcome.

NK receptor NKG2D interacts with several virus or stress inducible ligands, including ULBP1 (NKG2DL1) and -2 expressed on target cells. Induction of wild-type p53, but not mutant p53, strongly upregulated mRNA and surface expression of ULBP1 and -2, but not other ligands. An intronic p53-responsive element was discovered in these genes. Coculture of wild-type, p53-induced human tumor cells with primary human NK cells enhanced NKG2D dependent degranulation and IFN-γ production by NK cells.  

In the Tumor Micro Environment (TME) IFN-γ is produced at various concentrations in response to numerous immune stimulants and highlights the need for more personalized, disease centric approach. Engagement of IFN-γ Receptor on distinct tumor stromal cells, induction of interferon stimulated genes, immune status of the TME, and IFN-γ concentration are recognized as critical determinants for IFN-γ-mediated outcomes. Notably, an appropriate antitumor concentration of IFN-γ has yet to be determined. Interestingly IFN-γ produced by NK cells is said to be an essential mediator of Angiotensin II inflammation and vascular dysfunction.

Pharmacological activation of p53 exerts a potent antileukemia effect on antitumor immunity, including NK cell-mediated cytotoxicity against acute myeloid leukemia (AML). Interestingly, orally administered DS-5272 (a potent inhibitor of MDM2 - promotor of p53 degradation) induced upregulation of CD107a and IFN-γ in NK cells but not in CD8+ T cells. Furthermore, coculture of NK cells with leukemia cells resulted in massive apoptosis. 

Findings strongly suggest an interaction between B7 (NK receptor) molecules contribute to a particular design of the inflammatory microenvironment including B7-H6 and PD-L1, for which therapy was enhanced by expanded NK autologous or donor cells. RNA transfections, into HeLa cells of p53 or BRCA1 intron1 Key Sequences (based on Codondex iScore's most significant mRNA-intron1 variations) caused several genes to be upregulated, +1500% above control including B7-H6 (NCR3LG1) ligand for NCR3 (Nkp30) NK cell receptor which, when engaged triggers IFN-γ release. NCR3 and soluble isoforms of Leukocyte Specific Transcript 1 may play a role in inflammatory and infectious diseases. 

Blockade of B7-H3 prolonged the survival of SKOV3 ovarian cancer cell, an in ovarian tumor-bearing mice, miR-29c improved the anti-tumor efficacy of NK-cell by directly targeting B7-H3. miR-29c downregulates B7-H3 and inhibits NK-cell exhaustion. Low levels of mir-29c have been associated with mutated p53 in BC patients. miR-29 miRNAs activate p53 by targeting p85α and CDC42 and upregulate p53 levels that induce apoptosis in a p53-dependent manner. miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting IFN-γ. 

Besides (intron predominant) human ALU repeats, reverse complementary sequences between introns bracketing circRNAs are highly enriched in RNA editing or hyper-editing events. Knockdown of double stranded RNA-editing enzyme - ADAR1 significantly and specifically upregulated circRNA expression. In its absence (interferon stimulating) oligoadenylate synthetase (OAS) can be activated by self-dsRNA (in contrast to viral dsRNA), resulting in RNase L activity and cell death. Conversely, OASL1 expression enhanced RIG-I-mediated IFN induction. In cells absent of p53, immunogenic, endogenous mitochondrial dsRNA are produced and processed by the OAS/RNase L system presenting a novel mechanism in diseases with aberrant immune responses. IFN-γ restores the impaired function of RNase L and induces mitochondria-mediated apoptosis in lung cancer. The p53—OAS axis, in mitochondrial RNA processing may prevent self-nucleic acid such as dsRNA from aberrantly activating innate immune responses.

A plethora of evidence supports bottom up approach to personalized therapy. A p53 intron1-mRNA regulatory loop, as a potential mechanism in IFN responses to infection and disease may be diagnostic. Pre-clinical research, presently underway will establish whether p53 is diagnostic for specific selections of a biopsy to educate NK cells and trigger effective 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. 

Natural Killer to Kill or Transform?

Natural Killer cells emerge from distinct sources in embryonic development and each source of these earliest, innate immune cells confers different functions to the ascending cell lines. Fetal yolk sac, erythro-myeloid progenitor (EMP) derived NK cells, are uniquely biased for cytotoxic degranulation as opposed to inflammatory cytokine production, which are the dual hallmarks of all adult NK cells. But, parallel studies using human pluripotent stem cells (hPSCs) revealed that these progenitors can also give rise to NK cells that harbor a potent cytotoxic degranulation bias to kill.

In one of my previous articles, Natural Killer Shaping A Life I began to track processes supporting the unified origin of reproduction and immunity based on the theory of allorecognition.

Preceding implantation, the zygote divides to 16 cells that differentiate into an outer cell layer, trophoblast, and inner cell mass, embryoblast. The trophoblast becomes the fetal portion of the placenta, the embryoblast the embryo. Once differentiated into 30 cells a fluid-filled central blastocyst cavity forms. At about the 6th day of development, once it has reached nearly 100 cells the blastocyst mass begins its journey through the uterus to implant in the endometrium, which is where the embryo develops.

Yolk Sac

Implantation of the blastocyst is dependent, in part on a feto-maternal, immune handshake in which maternal NK cells of the Decidua (dNK), lining the Uterus are coerced by invading villus trophoblast cells to express cytokines that transform epithelial vascular cells in the placenta to release their binding enabling trophoblasts to replace them and  connect embryo to maternal blood supply  without rejection. This delicate phenomena is responsible for successful pregnancy. 

Following implantation, once cells of the inner embryoblast mass differentiate, only the cells of the yolk sac ultimately become the source of the NK cell of interest here. The other progenitor NK cell is sourced external to the yolk sac and that may also be basis of their functional differences.

The varied function of these dual sourced NK progenitors is transformation, as evidenced in trophoblast invasion, or killing damaged cells by cytolysis which the name "Natural Killer cell" describes. The dual source of the "killer" variety is thought to impart a WNT signaling influence over NK cell lineage. WNT-independent (WNTi-) and WNT-dependent (WNTd-) processes were found to distinguish the NK ontology. NK cells, biased toward degranulation and cell killing have been traced to the yolk sac, which remains isolated from rapidly differentiating external cells of the embryo until, over multiple weeks it gets fully absorbed.

dNK cells play an essential role in tissue and blood vessel transformation of the developing placenta. This has promoted an advanced body of thought that suggests certain tissue resident NK cells that possess some dNK characteristics may also be responsible for blood vessel transformation to accommodate new cancer or cancer stem cells that require new blood supply to develop into tumors. Therefore, cancer resident NK cells that, like dNK also express HLA-G or possess other trophoblast stem cell like characteristics may transform epithelial cells lining blood vessels in similar ways that connect fetal cells to the maternal blood supply without immune rejection.

To determine whether NK's varied ontology can shed any light on cancer cells coercing NK cells for tumor development and expansion, we identified the only three major gene expressions that are distinguished by their WNTi origin and which may inform about NK in adult mechanisms. Since our interest at Codondex is centered on p53 we correlated it with these genes:

1) NFIL3/E4bp4 transcription factor controls the commitment to NK lineage, directly regulates Eomes and ID2, which is responsible for P53 gain of function by suppressing ID2.

2) NCAM1/CD56 common NK gene is widely used to distinguish NK populations and strongly associated with p53 in multiple myeloma.

3) XBP1 a WNTi specific gene - regulates the p53/MDM2/P21 axis and is strongly present in yolk sac transcript analysis.  Unconventional splicing of XBP1 mRNA occurs in the unfolded protein response.

HLA-C is the only NK signaling molecule expressed by trophoblast cells that by a polymorphism can present variation for Adaptive immunity. Reduction of NK cytotoxicity was directly tied to the volume of Ets dependent expression from the site of the HLA-C polymorphism.  Further, Ets1, which is expressed in all NK ontologies, is necessary for a CBP/p53 transcription complex and transcription in UV-induced apoptosis in embryonic stem cells where the absence of p53 resulted in a high rate of embryonic malformations.

A possible scenario begins to emerge that near fully cytotoxic NK cells that bind HLA-KIR inhibiting and activating target cell receptors may be coerced to transcribe HLA-C, to express more inhibiting polymorphic or activating non-polymorphic transcripts, therefore greater or lesser Ets1 availability to coregulate XBP1 targets or CBP/p53 as the tipping point of degranulation and target cell killing.

Impotent Natural Killers by Cancer Stem Cells and Ageing

Cancer stem cells have been found, through various mechanisms to alter the sentinel function and innate, immune surveillance of Natural Killer cells (NK). In senescent cells that have stopped cell division, including in cancer stem cell niches and NK induced vascular remodeling (as found in the developing placenta) NK's sentinel vigilance is also reduced.

Senescence-associated mitochondrial dysfunction, a significant trigger of multiple dimensions of the senescent phenotype is caused by disruption of normal mitochondrial autophagy (mitophagy). Mitophagy increases with aging and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Deletion of a p53 response element on PINK1 promoter impacts p53-mediated PINK1 transcriptional repression. This p53-mediated negative regulation of autophagy has been found to be PINK1-dependent and constitutes a p53-PINK1 loop in nucleus and cytoplasm.

Further, mitophagy controls the activities of tumor suppressor p53 to regulate, at least hepatic cancer stem cells via Nanog. Prostate cancer cells escape NK attack by Nanog down-regulating ICAM1 (LFA1), to which NK would normally bind its target. In lung cancer NK have been found to limit the efficient clearance of senescent tumor cells from the mouse lung after p53 restoration. This indicated p53 may promote conditions for cellular survival and NK induced vascular remodeling or angiogenesis, necessary for the growth of tumors.

When under stress and inner mitochondrial membrane pressure gradient moves toward depolarization, Pink1 slots into the membrane, binds and phosphorylates p53 at Serine 392 (p53s392) and aids phagophore formation to enhance mitophagy. Mitophagy traps cytoplasmic p53s392, which reduces its transport to the nucleus where it would otherwise disrupt transcription of Nanog. (As illustrated below). 

Activated p53s392 nucleoside concentrations are effected by mitophagy

On the other hand, the sentinel function of NK may be subject to this PINK1 mediated mitochondrial switch. In prostate cancer cells Nanog promoted ICAM1 transcription required for NK binding target and cell killing. In prostate cancer cells Nanog over-expression restricts ICAM1, which promotes tumor formation. (As illustrated below). Investigating further, the direct functional link between p53 and ICAM-1 (CD54) in senescence and age-related disorders appears to be deeply integrated in mitophagy, senescence and immunity.

Nanog over-expression appears to be deterministic 

In stem cells where normal expression of Nanog transcribes ICAM1 and cancer stem cells where over-expression of Nanog restricts ICAM1, the variable PINK1-p53 switch may represent a "canary" that signals the state of  mitochondrial health to sentinel NK. However in some cancer cells where normal mitophagy is impaired and Nanog expression is restricted by p53s392, other p53 isoforms may directly promote the transcription of ICAM1.

In  two manipulation experiments using five different fibroblast cell lines that accelerated development of senescent associated secretory phenotypes a striking result was observed: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated pro-malignant paracrine signaling activities. Experiments show that PINK1 and Parkin, which are regulated by p53 specifically regulate mitochondrial antigen presentation of both MHC classes.

So, the question is whether the p53-PINK1 mitochondrial switch acts as cell-health "canary" for sentinel NK, where its inherent variables and regulatory loop may be fertile ground for the challenges of developing cancers? 

Therapeutic Coding and non-Coding DNA Relationships

Relationships of coding and non-coding intra-gene DNA are good cause for intense research and scientific debate. Many cellular functions of non coding DNA have been discovered in the past 30 years, but prior to that these genomic regions were mostly considered 'junk'.

Probing relationships between a genes' protein coding, cDNA and at least one non-coding DNA section of the transcript, which in our work is intron1 can yield important data about genomic features in the combination. Over the past 7 years we focused on interrogating combination relationships, across multiple transcripts to construct intra-gene DNA signatures from apparently disparate DNA elements that are known to perform vastly different biological functions, yet are proximal and often adjacent.

First we considered codon to amino acid coding may operate a little different to the classical view if reading a first and second nucleotide made the third deterministic. This method would not alter the outcome of known protein coding, but it may alter the way we consider combination relationships between nucleotide's. For a transcript, any given length of cDNA and its respective intron1 sequence could possess undiscovered intrinsic order. In a model where order was tightly honored, transcript relativity may identify cDNA sequences that caused significant change in the order at each next nucleotide step.

To investigate transcripts, from the first nucleotide we computed every length cDNA k-mer. We associated k-mer's, of every possible length with the cDNA transcripts intron1 signature. Then, for a set of multiple same gene transcripts, in nucleotide order our algorithm ordered the transcripts into a vector based on their respective cDNA-kmer:intron1-signatures. Stepping through from one k-mer to the next we observed whether next k-mer significantly changed the order of transcripts in the vector. After filtering domino effects we ranked k-mers with the most significantly changed transcript order from the previous k-mer.  

Size of  circle 'K' in the example indicates k-mer length, but we only compare same length K

In the above example, it is evident that k-mer2 vs k-mer3 was the most changed because all three transcript positions moved without a domino effect. From the vector we identify intra:inter transcript conditions in next nucleotide relationships as represented in the k-mers. 

As an example, in our work with 15 viable consensus transcripts for p53 occasionally all 15 transcripts in the vector changed positions at the next k-mer. These intra transcript k-mer relationships govern the transcripts order in the vector, but when, at the next k-mer transcript order is relaxed and positions move, particularly where the significant majority of positions move it is indicative that the intra transcript k-mer condition is relative to other transcript k-mers in the vector. The more and the further transcripts move positions in the vector the more relevant their intra transcript k-mer relationships are likely to be to gene.

This transcript comparative presents a new method for diagnosis and therapy because each new transcript, when compared to the consensus set has the capacity to disrupt order in the vector and yield k-mers that are specifically relevant to the gene. In our assay testing we were able to predict and synthesize ncRNA sequences that significantly reduced proliferation of HeLa cells. In our pre-clinical work, based on comparisons to transcripts of the TP53 consensus we will be predicting the efficacy of cell and tissue selections that educate and activate Natural Killer cells.

Pre-clinical flow chart to educate NK cells with tumor tissue/cell co-cultures and prove prediction

  

Synapses By p53 And CD40L in Reproduction and Immunity

Cell membranes constitute a diverse range of lipid molecules each attached to a varying, odd or even length hydrocarbon chain (a tail) that, collectively pack together to form a membrane. Packing is a dynamic that generally occurs according to surrounding pressure, concentration, hydrophobic conditions and motion. The mix of molecules and their hydrocarbon chains in each membrane play a crucial role in determining functions of complex organisms in cells.

Two complex membrane bound organisms of eukaryotic cells are mitochondria - primary provider of ATP energy powering reactions of the cell and endoplasmic reticulum (ER) - protein folding organelle surrounding the nucleus. The mitochondria comprise a double membrane containing electron transport chains - sets of four membrane bound proteins which pump protons between inner and outer membranes to maintain optimal inner mitochondrial membrane pressure through which oxygen is metabolized into water by phosphorylation of ADP to ATP molecules, which are the basic energy unit of the cell.

ER is a convoluted extension of the nucleus membrane into which translated amino acids are transported and where they fold before being released and packaged in the golgi apparatus and cytoplasm. The process of translation, folding and transport requires significant energy as such mitochondria and ER are closely associated. Recently and for the first time C18 ceramide transportation between ER and outer mitochondrial membrane was described as a cellular stress response mechanism.

Another important membrane lipid C16-ceramide was found to tightly bind within the p53 DNA-binding domain. This interaction was highly selective toward the C16 ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. This binding stabilized p53 and disrupted its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of downstream targets. The p53-MDM2 axis has been extensively covered in previous articles describing allorecognition, reproduction, immunity and auto-regulation. Ser241 was the only residue that interacted with all three p53 DNA sequences (p21, puma and a non-specific DNA system) persistently, indicating that Ser241 is a [response element] sequence-independent H-bond donor/acceptor for DNA.

It was also determined that Folate stress induces apoptosis via p53-dependent de novo Ceramide synthesis and up-regulation of Ceramide synthase 6 [C16], which is a transcriptional target of p53. In particular, Folate metabolism affects ovarian function, implantation, embryogenesis and the entire process of pregnancy. We observed that folate withdrawal leads to CerS6 up-regulation and C16-ceramide accumulation in a p53-dependent manner as a pro-apoptotic cue.

It has been demonstrated that clustering of the CD40 receptor depends on reciprocal clustering of the CD40 ligand, which is mediated by an association with p53, a translocation of acid sphingomyelinase (ASM) to the cell membrane, activation of the ASM (enzyme for ceramide), and a formation of ceramide. Ceramide appears to modify preexisting sphingolipid-rich membrane microdomains to fuse and form ceramide-enriched signaling platforms that serve to cluster CD40 ligand. Genetic deficiency of p53 or ASM or disruption of [C16] ceramide-enriched membrane domains prevents clustering of CD40 ligand. If the ligand is membrane-bound, the contact site between clustered ligands and receptors forms an immune synapse.

Finally, immune activation during the implantation phase causes preeclampsia-like symptoms via the CD40–CD40 ligand pathway in pregnant mice. The CD40 ligand (CD40L) is expressed by T cells and has a critical role in immune system regulation. Interventions targeting CD40L interactions following embryo implantation represent an approach to preventing preeclampsia (PE).

Here we have demonstrated a relationship between p53, C16 ceramide in reproduction and immunity via CD40 receptor-ligand in membrane bound concentrations of cells, particularly in respect of immunological synapse formation and blastocyst implantation. This further supports the notion that immunity and reproduction share common innate origins linked by p53.

A p53 Checkpoint For Cancer Therapy

Enormously complex signaling exists in the communication of antigens, receptors and ligands in DNA pathways between Natural Killer cells (NK) and target cells with which they interact. Based on observations, following NK formation of an immune synapse with its target cell two outcomes occur most often, termination or differentiation. The innate immune system comprises multiple cell types that are present and differentiated in tissues, but the predatory-like activity of NK has led to the general perception of its role in the immune system's front line.

As we have articulated many times on this blog, immunity and reproduction are tied, originally through allorecognition to the conserved p53-mdm2 axis. Further, it has become abundantly clear that auto-regulation of p53 occurs in multiple gene positive and negative feedback loops including mdm2. NK performance in young versus older patients showed a reduced capacity for, synaptic polarization and perforin release into the immune synapse before killing target cell. Further that the reduced release of perforin also reduced the capacity for NK to clear senescent cells associated with aging.

The activation of mitogen‐activated protein kinases (MAPK) is critical for lytic granule (perforin-granzyme) polarization, granule exocytosis and NK Cytotoxicity. It is possible that these proximal signalling events are compromised by aging. In addition, the studied p53 mutants regulated MAP2K3 gene whereas ectopic expression rescued the proliferative defect induced by mutant p53 knockdown.

In one series of experiments it was shown that the mutational status of p53 can facilitate cytotoxicity and different T cell recognition patterns. The p53 protein is presented by MHC molecules and the differential T cell recognition patterns seem confined to p53 as an antigen. The paper suggests p53 may behave differently to other classical tumor antigens, therefore a biomarker for immunotherapy targeting p53 should be the type of mutation expressed rather than protein levels only.

As previously reported, cytoskeleton superfamily member Talin1 has been uniquely tied to two essential NK functions;  activation of LFA1, required for binding ICAM on NK target cell and NK polarization that results. We know overexpression of talin head activates LFA1 and talin1 promotes cell proliferation by affecting the expression of BCL2 family and p53 network. But, mdm2 the conserved nemesis of p53 is neutralized by Merlin, another cytoskeleton superfamily protein also required for polarization. p53 also regulates the highly conserved Cdc42 which effects adhesion, actin cytoskeletal dynamics and cell movement including for angiogenesis in developing tumor microenvironments.

We found that activation of p53 augmented NK cell-mediated cytolysis of tumor cells via induction of ULBP2 expression on tumor cell surface. Further, we identified p53 as a direct transcriptional regulator of ULBP2 via an intron1 binding site, thus revealing previously unknown molecular mechanism controlling NKG2D ligand transcription. In mouse NK cells, talin is required for outside signaling by LFA1, which together with signaling by NKG2D induces granule polarization.

The functions of p53 are inextricably linked to multiple mechanisms in NK and target cells including recognition, antigen-receptor-ligand binding, cytoskeletal rearrangement, immune synapse, granzyme and perforin release. p53's mutation frequency and variances bearing p53 destabilizing mutations are recognized more effectively by p53-specific T cells than stabilized p53 mutants. Therefore, NK could operate its probe as a binding cipher that determines whether its target can be killed. Variable binding, and ectopic expression, resulting from a p53 feedback loop could be dependent on a p53 variable-kill-checkpoint that triggers the cascade of coordinated activities between NK and its target, generally referenced in the preceding paragraphs.

NK's p53 status, a targets MHC molecules presenting p53 antigens, ULBP2-NKG2D binding and relevant pathways confer with observations that the period of NK engagement is sufficient to allow downstream DNA transcription and translation to confirm and enable the kill event. Co-culture methods that could educate NK to better synchronize with targets, based on p53 status may usher in new regimes for organic immunotherapy. The Codondex research teams at Precision Autology are progressing through pre-clinical research using their computed cell selections.

Hope for a p53 Autologous Natural Killer Cell Therapy

Natural Killer Cells (NK) are much more than cell killers! They possess mechanisms and sensitivities that, among many functions, enables them at the front line of reproduction to interact with incoming trophoblasts that invade the uterine wall where NK cells are critical for blastocyst implantation and pregnancy. NK are members of the innate immune system, but they can be licensed to kill and re-purpose cells whereas most innate immune cells directly target invading pathogens.

Maternal decicdual NK may be redirected by PreImplantation Factor (PIF) expressing, anti-apoptopic, extra-villous trophoblasts that invade the endometrium (epithelioid) of the decidua of the uterine wall. This may result from epithelial LIF expression, and LIFR(eceptors) critical for blastocyst implantation. LIF allele's may act as a NK switch, the direct result of a p53 promoter allele that targets specific LIF transcription, that alters NK interactions with trophoblasts, the host endometria and vascular epithelia. If so, redirection of NK is an essential mechanism of conception that underwrites the development of the placenta.

Studies have revealed p53 targets LIF and demonstrated that, as a secreted protein LIF can function through the Stat3/ID1/MDM2 pathway to negatively regulate p53. Selected alleles in SNPs in LIF, Mdm2, Mdm4, and Hausp genes, each of which regulates p53 levels in cells, are also enriched in IVF patients. This association of SNPs in the p53 pathway with human fertility strongly suggests that p53 regulates human reproduction. It is distinctly possible enriched SNP's invoke regulation that negatively affects p53 and may also be the mechanism by which NK switches between modes that kill or transform its cell targets. In implantation, levels of p53  may lead to pre-eclampsia a condition that is the direct result of increased, p53 dependent apoptosis in extra-villous trophoblasts.

Pathogen-associated molecular pattern–mediated metabolic reprogramming can be considered as a manifestation of innate immune signaling, reprogramming a conserved phenomenon, that changes how we think about the biology and function of the innate immune network.

The mode of NK, in response to cancers may determine the fate of its target either by the binding of innate receptor combinations that initiate an immune synapse and perforin-mediated cytolysis or the release cytokines and chemokines that alters the inflammatory response. It was recently demonstrated these combinations are varied by different tissue and disease depending on p53 for example, in lung adenocarcinoma NK limited target killing and reduced inflammatory response allowing the cancer to spread. Further, peptides derived from p53 are presented by class I MHC molecules and may act as tumor-associated epitopes which could also be targeted by p53-specific T cells.  Results show that selected p53 mutations altering protein stability can modulate p53 presentation to T cells, leading to a differential immune reactivity inversely correlated with measured p53 protein levels.

These complex tissue dependent modes, through p53 pathways that contribute negative or positive feedback loop's, have prevented the most mutated gene in cancer from itself becoming a target of drug or immune therapy. Using a novel approach Precision Autology's Codondex algorithm computed the variable state of p53 isoforms, using a relative vector distance, from the consensus, to select patient cells for co-culture with, at least autologous NK for use in customized therapy. The approach will enable approved labs to identify highly specific cell targets, in part by their p53 state and to educate autologous NK cells based on a single p53 measure so that NK precision can be calibrated via the mismatch of target receptor combinations and p53.

Natural Killer Shaping A Life

As explained in previous posts, reproduction and innate immunity conspire when maternal Natural Killer (NK) cells of the decidua, lining the uterine wall are coerced to attack maternal epithelial cells, lining spiral arteries that penetrate the decidua to supply nutrients into the rapidly forming fetal placenta. The culprit, extravillous cytotrophoblasts that originate from the external wall of the blastocyst, penetrate the decidua and replace disrupted maternal epithelial cells of advancing spiral arteries. This rejection paradox by the maternal innate immune system, of the foreign male contribution to the blastocyst is mitigated by its trohphoblasts that enable maternal-fetal interface and blastocyst implantation. By day 7 life begins, at least through the handshake of maternal epithelial cells and fetal trophoblasts thus transforming rejection to inception.

Maternal NK enable extravillous cytotrophoblasts to converge with epithelial cells of spiral arteries

Decidual NK constitute 70% of lymphocytes up to the first 20 weeks of pregnancy. They are characterized by their low cytotolytic capacities, but adequately secrete cytokines, chemokines and angiogenic factors. As of 2018 it was unknown as to the effect of these decidual NK cells on earliest stages of pregnancy or how they may transform in context of the developing placenta. As previously discussed allorecognition by decidual NK cells is emerging as the key maternal-fetal immune mechanism that ultimately regulates placentation and that immuno-metabolism played a more significant role in NK activation and cellular transformation.

Single cell analysis at the Fetal - Maternal interface 

Studies of maternal microchimerism suggest that cell's and DNA transferred from mother to embryo can be traced and are prevalent in chord blood. These include NK cells that have been demonstrated to persist following re-transplantation of chord blood. Inferred in these findings, maternal microchimerism's, specifically NK cells transferred at a very early, even in single cell quantities may influence the earliest development of fetal immunity. Indeed at 6 weeks the earliest fetal NK cells are detected in the liver and tend to possess lower lytic potential a characteristic similar to decidual NK.

Maternal decidual NK cells that transfer into the developing placenta probably remain less cytolyic. Given the active environment they may even be metabolically exhausted, but are still capable of lytic activity and could play a critical role eliminating aberrant cells of the rapidly developing embryo. Further this activity could also educate fetal NK cells that start to develop from 6 weeks. Because this exposure occurs during early development of the fetal immune system, the primary response is to develop allospecific tolerance to maternal antigens.

A new concept is emerging in that the uterine immune system uses NK cell allorecognition to regulate placentation and to control the maternofetal interface. The jury is still out on microchimeric influences including exosomes, DNA and whole cells that transfer between mother and fetus. However, it seems entirely plausible that maternal immune cells may do much more than we presently know to shape conditions and determine cells of the fetus.

Our research interest relates to p53 peptides presented by MHC class receptors on targets of NK cells. We maintain the well conserved phospho-acceptor sites of p53 protein in axis with MDM2 is central to immunity and allorecognition. It is known that p53 plays an important role in blastocyst implantation and maternal reproduction through regulation of leukemia inhibitory factor (LIF) in mice. We expect p53 peptides, influenced by transcription regulatory factors determine outcomes of immune-target reactions including blastocyst implantation. Further that TP53 transcription can be triggered in a target by NK allorecognition nano-probe at a distance resulting in target p53 peptide presentation by MHC as NK's go-no-go cytolytic tipping point for immunity.