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.

Custom Immunotherapy To Address Dimorphic Complexities.

Dimorphic relationships between genes on Chromosome (Chr)6, encoding Human Leukocyte Antigens (HLA) and those on Chr19, encoding Killer-cell immunoglobulin-like receptors (KIRs) may eventually uncover important information as to how, why and when Natural Killer (NK) cells determine self restraint or attack cells infected by pathogens and disease. These proteins emerge from their respective zones, on each chromosome that have and continue to be subject to frequent recombination events.

The active region of Chr19 has a long history of recombinations that have and continue to define the expression patterns of telomeric and centromeric proportions of KIR gene's encoding receptors that bind cells presenting MHC class 1, HLA haplotype combinations that vary significantly across tissues in different population groups. Adding complexity, HLA genes on Chr6 are also subject to significant recombination making the dimorphic functional HLA-KIR interactions difficult to predict. 

Studies across population groups reveal the great diversity of HLA-KIR dimorphisms. The Southern Han centromeric KIR region encodes strong, conserved, inhibitory HLA-C-specific receptors, and the telomeric region provides a high number and diversity of inhibitory HLA-A and -B-specific receptors. In all these characteristics, the Chinese Southern Han represent other East Asians, whose NK cell repertoires are thus enhanced in quantity, diversity, and effector strength, likely augmenting resistance to endemic viral infections.

One study goes much further suggesting that functional interactions between KIR and HLA modify risks of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC) and that KIR B haplotypes provide selective pressure for altered p53 in BCC tumors. This preference implicates multi-modal p53 mechanisms that are also known to upregulate NK ligands, induce HLA-A11 assembly against Epstein Bar Virus and bind a frequently mutated p53 peptide in a complex with HLA-A and presented at the cell surface that prevent T-Cell response. In support, 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.

In addition to KIR, adaptive NKG2C+ NK cells display fine peptide specificity selectively to recognize HCMV strains that differed by a single substitution in the HLA-E-binding UL40-derived peptide during infection. Distinct peptides controlled the degree of proliferation in synergy with pro-inflammatory cytokines. Viral peptides are known to augment inhibition at NKG2A. Conversely, NKG2A+ NK cells sense MHC class I downregulation more efficiently than KIRs. Thus, both receptor:ligand systems appear to have complementary functions in recognizing changes in MHC class I.

Polymorphic landscapes across HLA, KIR and NKG receptor repertoires coupled with receptor:ligand haplotype cross referencing makes it near impossible to predict therapeutic targets across the breadth of disease and disease combinations that affect populations. A recent KIR-HLA co-existence study of haplotypes in Breast Cancer patients and controls highlights this complexity. 

Genetic signatures that target discovery of desired cell functionality to select preferential cells/tissues from micro environments used to educate and license autologous or allogeneic NK cells may tease specific, finely tuned, intact receptor repertoires. Once licensing efficacy is reached, expanding NK cell populations and applying them to act upon previously unrecognizable cells of a patient becomes the next frontier of immune therapy. This is the exciting work presently being undertaken by researchers and staff working with Precision Autology using Codondex methodologies. 

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. 

  

 

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. 

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.

What does COVID-19 have to do with heart attack?

Ground Glass Opacity's in Lungs

Natural Killer (NK) cells are depleted, but neutrophils are elevated in the lungs of hospitalized Coronavirus patients the world over. This is the sign of immune system chaos that typically precedes disease progression. How COVID-19 generates this condition is unknown, but surviving NK cells express NKG2A inhibitory receptors and are exhausted.

Typically patient CT scans reveal "Ground Glass Opacity's", fuzzy areas in lung scans that identify affected tissues. Without recovery at this point, the disease advances, tissues of the lungs can become infected, pneumonia may set it and soon after the patient may die.

It was recently published by doctors at Northwestern and UCLA that in around 20% of COVID-19 cases Troponin enzyme was elevated and correlated with very high mortality rates. Troponin is almost exclusively expressed in heart attack patients, so what does it have to do with COVID-19?

A different study tracked NK cells in lungs and linked pulmonary inflammation with depleted NK cells and elevated neutrophils. It found pulmonary NK cells control neutrophil intravascular motility and response to acute inflammation. Intriguingly, in a model of experimental myocardial infarction, NK cell depletion resulted in increased neutrophilic pathology in the lungs of mice, raising the question of how this influence is mediated. The study failed to identify the function of Ly6G, which by June 2019 remained unclear to the scientists what role it might play in the transfer of information between NK cells and neutrophils. A different team recently published a joint report showing lymphocyte antigen 6 family member E (Ly6E) impaired Coronavirus fusion and conferred immune control of viral disease. The link to Ly6 in these different reports may be important to front-line teams working to identify treatments.

In 2013 a joint China-Japan team had already published; "Lung Natural Killer Cells Play a Major Counter-Regulatory Role in Pulmonary Vascular Hyperpermeability After Myocardial Infarction". The report documented the counter-intuitive action of NK cells in lungs of mice induced with heart attack. Similarities to the reported behavior of NK cells and neutrophils in late stage COVID-19 patients expressing Troponin is remarkable. The team rescued the respiratory phenotype in NK cell–depleted mice by the adoptive transfer of NK cells from wild-type mice, but not from IL-10 knockout mice. All this may explain why preliminary successes have been achieved treating patients with plasma transfers or from patients who had recovered from Corona or with Mesenchymal stem cells.

Ly6 is only present in mice, but human neutrophils express the structurally related Ly6G molecule CD177 (19q13.31), a member of the Ly6/uPAR (urokinase plasminogen activator receptor) family. Interestingly, antibodies against CD177 have been shown to inhibit neutrophil transmigration across an endothelial monolayer, potentially by interfering with an interaction between Ly6G and PECAM1.

One interesting approach, at the right dosage may be to deploy a broad anti-venom aimed at 3TFx toxins because of their close resemblance to COVID-19 and Ly6 protein structures especially at the S1-CTD contact point. If anti-venom performs anywhere close to COVID-19 binding or connector domains it may impede it's entry to cells.

HCoV binding ACE2

A scientific conflict is brewing over the use ACE receptor inhibitor Captoptril against COVID-19 binding ACE. The drug is an angiotensin-converting-enzyme inhibitor derived from a peptide discovered in the venom of the lancehead viper (Bothrops jararaca). This debate over ACE2 upregulation may be preventing its broader use in patients despite its potential to reduce the capacity of COVID-19 to bind cells.

Its well known that several toxins and venoms can also lead to heart muscle injury, which COVID-19 seems to be emulating. The combination of viral immune response and false toxin signalling, that raises Troponin levels seems sufficient to trigger the immune system chaos that precedes typical disease progression and self-induced (possibly autoimmune) infection.

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.

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.