p53 Direct Mechanisms In Immunity

Never in the field of molecular oncology have so many sites of posttranslational modification in one protein (p53) been modified by so many different enzymes, but direct response mechanisms that increase immune receptors are rarely discovered and have important implications.  

In the tumor microenvironment (TME), cancer associated fibroblasts (CAFs) display an activated phenotype and can physically remodel the extracellular matrix (ECM). Silencing p53 in the CAFs strongly compromised this activity, implicating p53 as a key contributor to a distinctive CAF feature. Here, the non-autonomous, tumor-suppressive activity of non-mutant p53 cDNA is rewired to become a significant contributor to the CAFs’ tumor-supportive activities. This surprising role for p53 in CAFs suggests that, during tumor progression p53 functionality is altered, not only in the cancer cells, but also in their adjacent stroma.

Although p53 is not mutated in the human placenta, it has become functionally incompetent. Why and how p53 is functionally incompetent in cytotrophoblast cells might well be the key to understanding trophoblast invasion. Vascular remodeling for placentation is controlled by small populations of conventional Natural Killer cells, distinct from much larger populations of uterine NK cells, that acidify the ECM with a2V-ATPase, that activates MMP9, degrades the ECM and releases stored pro-angiogenesis growth factors. Similarly hypoxic TME's that in NK cells sustain excessive mitochondrial fission resulting in fragmentation could cause a2V-ATP activated MMP9 to similarly degrade ECM and promote angiogenesis in the early TME.  

Another MMP protein, MMP2 is a ligand for the Toll-like receptor 2 (Tlr2). Expression of Tlr2 and Tlr4 in the TME is important for the promotion of tumor growth, and when both of these receptors are absent, growth is compromised. Furthermore, the expression of Tlr2 and Tlr4 in both hematopoietic and stromal compartments appears to support MMP2-driven tumor growth.

The integration of the TLR gene family into the p53 regulatory network is unique to primates. p53 promoter response elements that are targeted by this DNA damage and stress-responsive regulator suggest a general p53 role in the control of human TLR gene expression. TLR genes show responses to DNA damage, and most are p53-mediated. TLR's mediate innate immunity to a wide variety of threats through recognition of conserved pathogen-associated molecular motifs. Expression of all TLR genes, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors with considerable inter-individual variability. Most TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites.

A polymorphism in a TLR8 response element provided the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings—demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress—have many implications for health and disease, as well as for understanding the evolution of DNA damage and p53 responsive networks. That p53 can directly increase an inflammatory response differs from the generally held view relating to the antagonistic affect of p53 on inflammation directed by NF-κB. However, the direct mechanism here is different in that it involves another p53-mediated increase in a receptor that translates ligand interactions into cytokine responses.

p53 - Mediator Of Natural Killer Education

The regulation of rapidly transforming stem cells into trophoblasts and expanding embryonic cell phenotypes, between gestation day 8 and 15 is fast and furious. Research unraveling the finer detail points to the advent of pressure impacting evolving conditions for growth, transformation of cells, microvasculature and resulting tissue types. Notably, Natural Killer (NK) cells expand to around 30% of the cells in the stroma of the uterine wall. These uterine NK (uNK) cell subsets coexist alongside conventional NK cells. This unusual uNK quantitative imbalance motivated our research.   

uNK are closely associated with spiral artery remodeling, for placentation at the blastocyst implantation site. They possess a functional Renin- Angiotensin system (RAS), the cornerstones of blood pressure. The ratio of uNK cells expressing Angiotensin II receptor type 1 (AT1) markedly changed between gestation day 6 and 10. At day 10-12 Atrial Natriuretic Peptide, for vasoconstriction and dilation, strongly co-localized to uNK cells at the implantation sites. Expression of these vasoregulatory molecules by uNK suggests they contribute to the changes in blood pressure that occur between days 5 and 12 coincidental with their population explosion in the decidua during normal pregnancy.

Similar to Angiotensin, Bradykinin (BK) is produced from an inactive pre-protein kininogen that is activated by serine protease kallikrein (KLK), mostly represented on chromosome 19, where they associate with a number of other genes involved in blood pressure. Oakridge scientists predicted that BK induced a Covid19 "cytokine storm" that is responsible for disease progression. 

KLK's are located at 19q13.41, an active transposon region with a 2x background deletion rate clustered near Zinc Fingers and KIR's (Killer immunoglobulin like receptors) that inhibit NK cells.  A link was confirmed in mice uterine NK cells that regulated local tissue blood pressure, by at least AT1, partly in response to mechanical stretch of vasoconstriction and dilation induced by uterine NK's internal RAS. 

In reproduction, at  Chromosome 19 MiRNA Cluster (C19MC), 59 known miRNAs are highly expressed in human placentas and in the serum of pregnant women. Numerous C19MC miRNA's are also found in peripheral blood NK's and at least miR-517a-3p (a C19MC from fetal placenta) was incorporated into maternal NK cells in the third trimester, and was rapidly cleared after delivery. miRNA's also regulate the migration of human trophoblasts and suppress epithelial to mesenchymal transition (EMT) genes that are critical for maintaining the epithelial cytotrophoblast stem cell phenotype. 

In hepatocellular carcinoma (HCC) a co-regulatory network exists between C19MC miRNAs, melanoma-A antigens (MAGEAs), IFN-γ and p53 that promotes an oncogenic role of C19MC and is disrupted by metal ions zinc and nickel. IFN-γ plays a co-operative role whereas IL-6 plays an antagonistic role. Its an important immunoregulartory network, because, in the very least, IFN-γ and IL6 have a major baring on the expression of HLA/MHC molecules on cancer cells. 

Immediately adjacent to C19MC, is the leukocyte immunoglobulin-like receptor complex, from where LILRB1 receptor, also known as Mir-7, is expressed on NK cells. It binds MHC class I molecules, on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB1 has a polymorphic regulatory region that enhances transcription in NK Cells and recruits zinc finger protein YY1 that inhibits p53. It is required to educate expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

In 2019 a study of arsenite-induced, human keratinocyte transformation demonstrated that knockdown of m6A methyltransferase (METTL3) significantly decreased m6A level, restored p53 activation and inhibited phenotypes in the-transformed cells. m6A downregulated expression of positive p53 regulator, PRDM2, through YTHDF2-promoted decay of mRNAs. m6A also upregulated expression of negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, the study revealed the novel role of m6A in mediating human keratinocyte transformation by suppressing p53 activation and sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.

In 2021 a discovery that YTHDF2 is upregulated in NK cells upon activation by cytokines, tumors, and cytomegalovirus infection. YTHDF2 maintains NK cell homeostasis and terminal maturation. It promotes NK cell effector function and is required for IL-15-mediated NK cell survival and proliferation by forming a STAT5-YTHDF2 positive feedback loop. Analysis showed significant enrichment in cell cycle, division, including mitotic cytokinesis, chromosome segregation, spindle, nucleosome, midbody, and chromosome. This data supports roles of YTHDF2 in regulating NK proliferation, survival, and effector functions. 

As part of the 2021 discovery, transcriptome-wide screening identified TDP-43 to be involved in cell proliferation or survival as a YTHDF2-binding target in NK cells. TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons. Growth of the developing cerebral cortex is controlled by Mir-7 through the p53 Pathway

Here we have broadly described mechanisms by which NK cells maintain tissue homeostasis where tightly regulated p53 optimizes cellular conditions to 'self' educate the expanded NK cells. Those that express NKG2A and/or one or several KIRs, for which cognate ligands are present, become educated and as such transform to potent killers in response to their missing-self. Therefore, p53 isoforms have the innate capacity to promote a cellular homeostasis that makes it the mediator for optimal education of expanded NK cells.

ΔΨm and Immune Responses to Disease

Each cell contains hundreds to thousands of mitochondria, each with hundreds of electron transport chain complexes (ETC) that deliver ATP as the cells primary energy source and the central dogma of eukaryote existence. ETC function's, on the inner mitochondrial membrane, are sensitive to change in electric charge represented as mitochondrial polarization and mitochondrial membrane potential (ΔΨm). The large responsive surface area of the outer and inner membrane promotes remodeling and protein interactions that may lead to cellular diseases including cancer.

Tumor Necrosis Factor (TNF) causes mitochondria to relocate, to bind the nucleus and efficiently shuttle elements that enable fast DNA transcription and signaling that, under certain conditions, may suppress the pro inflammatory immune response. TNF signaling to mitochondrial PINK1 stabilizes ubiquitin chains that result in mitochondrial relocation and shuttling activated p65 that increases NF-κB transcription in the nucleus. This anti-apoptotic response resembles the feed forward activation loop in Pink1/Parkin-dependent mitophagy as an independent defense against accumulation of dysfunctional mitochondria, that under physiological conditions integrate their roles in innate immune signaling and stress. 

Enhanced activation of NF-κB by TNF, via mutant p53, concomitantly suppressed the pro-apoptotic effect of TNF leading to increased invasiveness of cancer cells. Accordingly mutant p53 may directly affect nuclear accumulation and retention of p65 upon cytokine exposure as mutant p53 overexpression and nuclear p65 staining in tumors strongly correlated.

Stresses elicited by aneuploid states in cells mediate interaction between Natural Killer (NK) cells. In highly aneuploid cancer cell lines NF-κB signaling is upregulated and activated promoting immune clearance by NK cells, but anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. Rapid NF-κB signaling may be preferentially selected because it antagonizes p53, known to inhibit the growth of highly aneuploid cells. Significantly increased mitochondrial DNA in aneuploid cells may result from increased fission of mitochondria, similar to that found in extreme ploidy during Oocyte development. Perhaps supporting the reason in embryonic stem cells (ESC) apoptosis occurs independent of p53 and protein kinase Akt3, the regulator of ESC apoptosis, suppresses p53 for the survival and proliferation of these stem cells.

A comprehensive metabolic analysis identified mitochondrial polarization as a gatekeeper of NK cell priming, activation, and function. Mitochondrial fusion and OXPHOS promote long-term persistence and improve cytokine production by NK cells. Hypoxic Tumor Micro Environments (TME) sustained NK cell activation of mTOR-Drp1, which resulted in excessive mitochondrial fission and fragmentation. Inhibition of fragmentation improved mitochondrial metabolism, survival and the antitumor capacity of NK cells. 

Mitochondrial biogenesis also requires the initiation of Drp1-driven fission. Whereas, fissions from dysfunction are associated with diminished ΔΨm and Reactive Oxygen Species (ROS), which are unchanged in this biogenesis. Depletion of p53 exaggerates fragmentation, but does not affect ΔΨm and ROS levels. Instead, p53 depletion activates mTORC1/4EBP1 signaling that regulates MTFP1 protein expression to govern Drp1-mediated fission. Thus, increased fission upon p53 loss can stimulate biogenesis, but not accumulation of damaged mitochondria. This may explain how mitochondrial integrity, in context of p53 deficiency induced fragmentation, may suppress immune signaling.

Downregulating p53 expression or elevating the molecular signature of mitochondrial fission correlates with aggressive tumor phenotypes and poor prognosis in cancer patients. Upon p53 loss, exaggerated fragmentation stimulates the activation of ERK1/2 signaling resulting in epithelial-to-mesenchymal transition-like changes in cell morphology, accompanied by accelerated MMP9 expression and invasive cell migration. Notably, blocking the activation of mTORC1/MTFP1/Drp1/ERK1/2 axis completely abolishes the p53 deficiency-driven cellular morphological switch, MMP9 expression, and cancer cell dissemination. MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. 

Vascular remodeling, in the uterus, during pregnancy is controlled by small populations of conventional Natural Killer cells that acidify the extracellular matrix (ECM) with a2V-ATPase that activates MMP9, degrades the ECM and releases pro-angiogenesis growth factors stored in the ECM. Hypoxic TME's that sustain excessive mitochondrial fission-fragmentation in NK cells would cause a2V-ATP activated MMP9 to similarly promote angiogenesis akin to Blastocyst implantation.  

ΔΨm as a measure of functional integrity maybe the flawed alert, a blind spot for the 'canary in the mine' of a cells' ADP-ATP pipeline. Likewise the status of TP53, from transcription through p53 isoform, may signal wide ranging affects of ΔΨm that incorporate fragmentation, accumulating damaged mitochondria, mitophagy, apoptosis, normal immune signaling and response through to mitochondrial biogenesis, differentiation, angiogenesis, reduced immune signaling and response. This modal duality aligns known functions of NK cells that under physiological conditions promote angiogenesis growth (as in Blastocyst implantation and placental vascularization) or NK's classic, cytolytic role in the innate immune response. 

The delicate balance in health and sensitivity of at least TP53 DNA is known to result in DNA to DNA and/or upstream RNA/protein interactions that influence mechanics of molecules and responses to ΔΨm variations. Here we have highlighted links between NK cell function relative to  mitochondrial polarization, ΔΨm and p53 relative to mitochondrial fission and immune signaling. 

Life, Dormancy or Death?

Cellular biology is viewed through different lenses, but pregnancy offers a perspective on the invasive origin of cell division, the senescent state and cancer. Pregnancy causes Natural Killer cells of the decidua (dNK) to expand abundantly until they represent as much as 30% of the mucous membranes' cells. NK cells may be induced to expand by invading trophoblasts to realize the dNK trifecta - robust innate immunity that protects the embryo from maternal infection, modulation of trophoblast invasion and driver of vascular remodeling. However, in many cancers expansion of diverse NK populations fails to materialize and missing sub-sets of NK cell diversity provides a path for cancers unchecked growth. 

In decidual cells at the human maternal-fetal interface, CD82 - the metastasis suppressor may participate in intercellular communication with trophoblasts and limit their invasiveness. Trophoblasts enhance adhesiveness of dNK to the decidua's stromal cells, via the CXCL12/CD82/CD29 signaling pathway which contributes to CD56bright NK cell enrichment a necessary element for heathy pregnancy.

CD82 expression is downregulated in tumor progression of many human cancers and strongly correlated with tumor suppressor p53. It can be activated by p53 through a consensus binding sequence in the promoter. In human ovarian cancer a sequential genetic change at the TP53 and the CXCL12 receptors CXCR4  locus occurs during transformation of surface epithelium. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 was10-fold higher compared to that in parental HCT116 cells with functional wild-type p53.

The CXCL12 ligand is unique for its CXCR4 receptor and both are expressed in human first-trimester endometrial epithelial cells (EECs) at the mRNA and protein level. EEC-conditioned medium and recombinant human CXCL12 significantly increased the migration and invasion of EECs. CXCL12 has also been associated with the recruitment of CD56bright CD25+ dNK subsets in early pregnancy's.

CXCR4 is specifically upregulated in the human endometrium during the implantation window and increased immunostaining observed only when a blastocyst is present. CXCR4/CXCL12 not only enhances trophoblast invasiveness, but also limits over-invasiveness by upregulating CD82. CXCR4 activation increases the CXCL12-CXCR4 signaling axis stimulates vascular endothelial growth factor (VEGF) synthesis which induces CXCR4 and CXCL12 production. This synergistic regulation influences placental vascularization. CXCR4 suppresses apoptosis and increases the viability of trophoblasts. 

Undetectable disseminated tumor cells, in different tissue microenvironments restrain or allow the progression of breast cancer in the liver where in dormant milieu's there are selective increases in NK cells. Stroma crosstalk and exit from dormancy follows a marked contraction of the NK cell compartment and concurrent accumulation of activated hepatic stellate cells (aHSCs). Proteomics on liver co-cultures implicate aHSC-secreted CXCL12 in the induction of NK cell quiescence through CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases and were inversely correlated with NK cell abundance.

The dNK behavior that checks trophoblast invasion and promotes vascularization resembles immediate and invasive new cancers that may occur in cells of any tissue environment. Similarly expansion of resident tissue NK sub-sets in response may be the determiner of life, the shape of next generation cells, dormancy or death.  

Blood Pressure by Natural Killer Cells and SARS-CoV2

A meta-analysis of gene expression signatures for diastolic, systolic blood pressure and hypertension found that out of 7717 unique, related genes 34 were most differentialy expressed across 7017 individuals from 6 international studies. No less than 20% of the 34 gene's, were located on Chromosome 19. Enrichment analysis for the diastolic and systolic gene group's associated to Natural Killer (NK) Cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response, pointing strongly to innate and adaptive immunity. 

I covered the NK origin of MHC and antigen immunity and reproduction at Chromosome 19 on a previous blog, now the meta-analysis adds infection, immunity and blood pressure to this location. Evolutionary detectives tracked events from Chromosome 19 to 1,6 and 9 via transposon re-combination events, which provided further direction for interpreting the blood pressure meta-analysis. A review of the genes and pathways involved increasingly characterized innate immunity as an integrated core component of almost every aspect of our skeletal, circulatory, tissue and neuronal systems. 

Blood pressure is enormously complex, but its governance of entropy under the mechanical laws of molecular diffusion and disassociation reign supreme. Renin-Angiotensin (RAS) genes are widely recognized to be the cornerstones needed for blood pressure. Innate immune cells including NK have been confirmed to possess and express RAS genes. Macrophages, a member of innate immune system have been linked to angiotensin signaling neuropathic pain as well as bacterial infection inducing pain suppression by angiotensin 2 receptor (AT2R).  Maternal NK cells AT1R and AT2R have been implicated in the control of localized blood pressure in placental tissues leading to preeclampsia a condition in pregnancy.

In various studies, including in disease conditions it has been shown and suggested that different male:female ratios between AT1R and AT2R in monocytes and other innate cells is an important factor in the determination of blood pressure that has been extensively studied in heart and lung conditions. AT2R plays a critical role in satellite cell differentiation and skeletal muscle regeneration via myoblasts, which may be the reason it's expressed ubiquitously in developing fetal tissue. It's likely that balance between AT1R and AT2R signaling is critical for normal muscle regeneration.  

In addition to the role of NK cells in blood pressure a study using lung-intravital microscopy linked pulmonary NK cells to the control of neutrophil intravascular motility, response to acute inflammation and diminished pathogenic accumulation. NK cell derived IFN-γ plays an important role in the activation and maturation of monocytes into macrophages and dendritic cells, an amplifying mechanism in the early innate inflammatory response. Angiotensin II can induce rapid neutrophil infiltration via AT1R that also stimulates leucocyte–endothelium interactions. Inhibited IFN-γ signaling ameliorated Angiotensin II induced cardiac damage, which led to a finding that NK-cells play an essential role in the induced vascular dysfunction.

Ace2 Expression Patterns in Covid19

Pathophysiology of Covid19 demonstrates that NK cells are depleted and neutrophils infiltrate into lung tissue leading to tissue damage and escalation of the disease. By SARS-CoV2 binding the Ace2 receptor of vascular epithelial and other cells, the conversion of Angiotensin II is blocked (image above), therefore upregulated. Increased levels of Angiotensin II were shown to induce NK cells to release IFN-γ. On recruitment to inflammatory sites, NK-cells release IFN-γ and engage with monocytes in a reciprocal program of activation in which monocytes mature into macrophages and dendritic cells. NK exhaustion results and is a known outcome that may relate to IFN-γ levels. However, in patients with high expression of Ace2, NK cell counts are lower and cytokine expressions do not show up during the initial disease state pointing to the role of accumulating Angiotensin II.  

Covid19 Meta Analysis

The image above shows distribution of expression (y axis) for ACE2, PCSKs (blood pressure mediation) and TMPRSS2 (CoV2 S1 cleavage) across lung cell types (x axis). It completes the picture that Corin-Furin mediated control over blood pressure normalization is a significant component of Covid19 disease progression and NK cells are a central player.

Molecules targeting RAS are a major focus of inhibitory or complementary therapeutic design, but a modified NK cell that is shielded from SARS-CoV2 may be the tool-in-the-shed our immune systems need. 

Is The Natural Killer our Anti-Cell?

Lymphocyte

Natural Killer (NK) cells may be our innate, anti-cell sentinels that first arise in the yolk sac between day 8 and 10. Simultaneously maternal, uterine NK cells promote fetal development by secreting growth promoting factors. Distinct sets of NK cells mature during early fetal development by associating with differentiating cells and tissue environments. Typically NK cells mature in around 25-35 days. By comparison, the developing embryonic heart begins pumping blood around day 22. The transcriptome, lineage and variety of each terminally differentiated embryonic NK cell and whether, in adults they continue to exist is unknown and genetically undefined.

The prospect of innate immune memory is a developing interest that has been shown to link innate and adaptive cell sets by epigenetic responses. Natural Killer cells have been a particular focus of this research because they were once considered innate and non-adaptive. Recently there is an emerging body of evidence that suggests otherwise. Whether NK cell memory arises and which NK cells participate in its establishment is not established science. Without more research whether and to what extent very narrow NK cell protein expressions distinguish cell and tissue lineages will continue unanswered. However, some good research supports the important idea of NK memory and conserved lineage.

A study on the heterogeneity of NK cells, by transcriptome in human bone marrow identified distinct NK populations, including one expressing higher levels of immediate, early genes indicative of a homeostatic activation.  Analysis identified a transitional population between CD56bright and CD56dim NK cells. Most interesting they reported on a donor with GATA2-T354M mutation who exhibited a reduced percentage of CD56bright NK cells with altered transcriptome and elevated cell death indicating the smaller number of CD56bright NK cells were contributing to the donors disease progression.

In a mouse model of CMV infection, a specific population of NK cells expands, contracts after control of the virus and generates long-lived “memory” NK cells that are more protective during a second encounter. Other reports indicate antigen specificity and antibody dependent NK mediated cytotoxicity in autoimmune disease by a sub-population of NK cells expressing a combination of specific receptors that was associated with apoptosis and the depletion of IgG in individuals with autoimmune thyroid disease.

Coronavirus Lung Cells

In Covid19 admitted patients NK cells and lymphocytes were depleted including by apoptosis and exhausted. This late stage condition developed for some time prior to admission. The published research identified two receptors as particularly important for CoV2 viral entry into a cell. Ace2 to which CoV2 binds and TMPRSS2 that cleaves its Serine protein enabling entry. Only one report, so far provided useful information about expression of these genes in NK cells of lungs. From the 57,020 lung cells on the UMAP plot only NK cells expressing Ace2-TMPRSS2 connected Muscle, Fibroblasts, T-Cells and Macrophages.

A four year experiment tracked bar-coded NK cells introduced to rhesus macaque's. The results indicated that during homeostasis and moderate proliferative stress, peripheral, compartmentalized, self-renewal can maintain the composition of distinct, differentiated NK cell sub-populations.

Blunted categorizations of NK receptor expressions may be a sub-set limitation, but the good news is that more recent research is defining NK cells in increasingly diverse ways. We are also learning to identify diseases that infect NK cells including influenza, that induces apoptosis, HIV, Hepatitis c and Epstein Bar. A study found peptide specific recognition of human cytomegalovirus strains control expansion of adaptive NK cells. Another Covid19 study compared the transcriptome in lung tissues of older patients, including NK cells and concluded that genes induced by SARS-CoV-2 infection tend to increase in expression with aging, and vice versa.

Although still a little abstract for science, its plausible that a cell contracting a disease could recall its NK 'memory cell', its NK anti-cell counterpart to annihilate it because it differentiated beyond its NK anti-cell phenotype range. If true, an indiscriminate disease that also targeted the NK anti-cell and eliminated it would prevent annihilation of its phenotype differentiated counterpart cells. By example, SARS-CoV2 would also bind the Ace2-TMPRSS2 expressing NK anti-cell, infect and kill it then NK immune resistance would be compromised and cell differentiation may rapidly progress to become lethal.

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.

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.