All Roads Lead to (Ch)Romosome 19!

A hepatocellular carcinoma (HCC) co-regulatory network exists between chromosome 19 microRNA cluster (C19MC) at 19q13.42, melanoma-A antigens, IFN-γ and p53, promoting an oncogenic role of C19MC that is disrupted by metal ions zinc and nickel. IFN-γ plays a co-operative role whereas IL-6 is antagonistic, each have a major bearing on the expression of HLA molecules on cancer cells. Analysis of Mesenchymal stem cells and cancer cells predicted C19MC modulation of apoptosis in induced pluripotency and tumorigenesis.

Key, differentially expressed genes in HCC included cancer-related transcription factors (TF) EGR1, FOS, and FOSB. From mRNA and miRNA expression profiles these were most enriched in the p53 signaling pathway where mRNA levels of each decreased in HCC tissues. In addition, mRNA levels of CCNB1, CCNB2, and CHEK1, key markers of the p53 signaling pathway, were all increased. miR-181a-5p regulated FOS and EGR1 to promote the invasion and progression of HCC by p53 signaling pathway and it plays an important role in maturation or impairment of natural killer (NK) cells.

A pan-cancer analysis, on microRNA-associated gene activation, produced the top 57 miRNAs that positively correlated with at least 100 genes. miR-150, at 19q13.33 was the most active, it positively correlated with 1009 different genes each covering at least 10 cancers. It is an important hematopoietic, especially B, T, and NK, cell specific miRNA.

Rapid functional impairment of NK cells following tumor entry limits anti-tumor immunity. Gene regulatory network analysis revealed downregulation of TF regulons, over pseudo-time, as NK cells transition to their impaired end state. These included AP-1 complex TF's, Fos, Fosb (19q13.32), Jun, Junb (19p13.13), which are activated during NK cell cytolytic programs and down regulated by interactions with inhibitory ligands. Other down-regulated TF's included Irf8, Klf2 (19p13.11), Myc, which support NK cell activation and proliferation. There were no significantly upregulated TF's suggesting that the tumor-retained NK state arises from the reduced activity of core transcription factors associated with promoting mature NK cell development and expansion.

Innate immune, intra-tumoral, stimulatory dendritic cells (SDCs) and NK cells cluster together and are necessary for enhanced T cell tumor responses. In human melanoma, SDC abundance is associated with intra-tumoral expression of the cytokine producing gene FLT3LG (19q13.33) that is predominantly produced by NK cells in tumors. Computed tomography exposes patients to ionizing X-irradiation. Determined trends in the expression of 24 radiation-responsive genes linked to cancer, in vivo, found that TP53 and FLT3LG expression increased linearly with CT dose. 

Undifferentiated embryonal sarcoma of the liver displays high aneuploidy with recurrent alterations of 19q13.4 that are uniformly associated with aberrantly high levels of transcriptional activity of C19MC microRNA. Further, TP53 mutation or loss was present with all samples that also display C19MC changes. The 19q13.4 locus is gene-poor with highly repetitive sequences. Given the noncoding nature and lack of an obvious oncogene, disruption of the nearby C19MC regulatory region became a target for tumorigenesis. 

The endogenous retroviral, hot-spot deletion rate at 19p13.11-19p13.12 and 19q33-19q42 occurs at double the background deletion rate. Clustered in and around these regions are many gene families including KIR, Siglec, Leukocyte immunoglobulin-like receptors and cytokines that associate important NK gene features to proximal NK genes that were overrepresented in a meta analysis of blood pressure. 

Endogenous retroviruses that invite p53 and its transcriptional network, at retroviral hot-spots, suggest that lymphocyte progenitors, such as ILC's and expanded, NK cells are synergistically responsive to transcription from this busy region including by the top differentially expressed blood pressure genes MYADM, GZMB, CD97, NKG7, CLC, PPP1R13L , GRAMD1A as well as (RAS-KKS) Kallikrein related peptidases to educate early and expanded NK cells that shape immune responses.  

Indispensable Mitochondria - Cancers back door?

Immediately prior to fertilization spermatozoa are devoid of Mitochondrial DNA (mtDNA), potentially explaining an aspect about selection that may serve the legacy for maternal immune tolerance. Post fertilization, on day 11-13, outermost trophoblasts of the blastocyst dock with the decidual lining as it embeds in the uterine wall. Then, maternal vascular remodeling and placental formation begin toward successful implantation. 

Higher quality trophoblasts are associated with lower mtDNA content. Moreover, euploid blastocysts with higher mtDNA content had a lower chance to implant and mtDNA replication is strictly downregulated between fertilization and the implantation. What is it about absent or reduced mtDNA that may also relate to the mechanics of immune tolerance and vascular remodeling, which are also features of solid tumors.

The initial absence or downregulation of MtDNA, may relate an immune tolerance by uterine Natural Killer (NK) cells. As mtDNA upregulates, after day 12, it may initiate NK auto-reactivity required for maternal microvascular remodeling. This auto-immune paradox is a prerequisite for vascular remodeling, which is also seen in localized hypertension, and the likely basis of successful blastocyst implantation. Acutely, micro-hypertension induced mechanical stretch, on endothelial cells, interconnects innate and adaptive immune responses. 

The dominant cell in the decidua is an NK subset (dNK), they express low levels of IFN-γ and express proteins of Renin Angiotensin System (RAS). At day 12 RAS peptide ANP colocalizes to dNK’s suggesting that dNK RAS infers localized responsiveness.  When TFAM, required for transcription of mtDNA, was deleted from cardiomyocytes, after 32 days, animals developed cardiomyopathy and Nppa (gene encoding ANP) and Nppb expression was elevated. 

In monocytes increased endothelial stretch activates STAT3, which is involved in driving almost all pathways that control NK cytolytic activity and reciprocal regulatory interactions between NK cells and other components of the immune system. The crosstalk between STAT3 and p53/RAS signaling controls cancer cell metastasis. p53, Stat3, and, potentially, the estrogen receptor are thought to act as co-regulators, affecting mitochondrial gene expression through protein-protein interactions. Co-immunoprecipitation of p53 with TFAM suggests it may regulate mitochondrial DNA-damage repair.

Like initial trophoblasts with low level mtDNA, mature cells, like cardiomyocytes that prolong low level mtDNA may also aggravate autoimmune sponsored hypertension that remodels microvascular networks providing nutrients for growth of reduced mtDNA stem cell replicas. Indeed, mitochondrial dysfunction (from depleted mtDNA) does not affect pluripotent gene expression, but results in severe defects in lineage differentiation.

During severe sepsis, intense, on-going mtDNA damage and mitochondrial dysfunction could overwhelm the capacity for mitochondrial biogenesis, leading to a gradual decline in mtDNA levels over time. This may contribute to monocyte immune deactivation, which is associated with adverse clinical outcomes and could be reversed by IFN-γ. 

Identifying cells that optimally educate cocultured NK cells for precision IFN-γ and cytolytic responsiveness is part of the ongoing work by the Codondex team.

Educating Perfect Natural Killers

Mining Tissue Match for Immune Co-culture

Mutant p53 knockdown in KPC (pancreatic ductal adenocarcinoma) cells of immune deficient mice had no effect on primary tumor growth, by contrast the reduced tumor growth in the immune-proficient syngeneic host was due to altered immune cell recruitment.

In vivo, the increased production of pro-inflammatory cytokines coupled with increased Natural Killer (NK) cell ligand expression permits the recruitment of immune cells and clearance of abnormal cells. Elimination of senescent tumors by NK cells may occur as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells.

Coculture of wild-type (wt) p53-induced human tumor cells with primary human NK cells enhanced NKG2D-dependent degranulation and IFN-γ production by NK cells. Taken together findings define the involvement of p53 in the regulation of specific NKG2D ligands that enhance NK cell–mediated target recognition.

Inhibitory KIR-educated NK cells showed significantly increased expression of the glucose transporter Glut1 in comparison to NKG2A-educated or uneducated NK cells, with and without exposure to target cells. Educated NK cells displayed significantly higher rates of cellular glycolysis than uneducated NK cells indicating they may reside in different metabolic states prior to activation. The ability to metabolize glucose may represent a mechanism for the superior functionality of educated NK cells expressing KIR receptors. 

Cancer cells acquire immunoediting abilities by which they evade surveillance and escape eradication. Murine p53 missense mutation G242A (human G245A) suppresses activation of host NK cells, enabling breast cancer cells to avoid immune assault. Serial injection of EMT6 breast cancer cells that carry wild-type (wt) Trp53 promoted NK activity, while SVTneg2 cells carrying Trp53 G242A+/+ mutation decreased NK cell numbers and increased CD8+ T lymphocyte numbers in spleen. Upon co-culture with isolated NK cells, EMT6 cells activated NK cells and proliferation, increasing interferon-gamma (IFN-γ) production; however, SVTneg2 cells suppressed NK cell activation. p53 can modulate expression by cancer cells of Mult-1 and H60a activating and inhibitory ligands for NKG2D receptors of NK cells, respectively, to enhance immune surveillance against cancer. p53 is requisite for NK cell-based immune recognition and elimination of cancerous cells, and p53 missense mutant in cancer cells impairs NK cell responses.

NK cells are the oldest member of the innate lymphoid cell family (ILC) and the only representative of cytotoxic ILCs. These tissue-resident innate immune cells have a similar functional diversity to T cells including lineage-specifying transcription factors that drive certain effector programs. ILCs are present in almost every tissue, but strongly enriched at barrier surfaces, where they regulate immunity to infection, chronic inflammation, and tissue maintenance. ILCs orchestrate tissue homeostasis through their ability to sustain bidirectional interactions with epithelial cells, neurons, stromal cells, adipocytes, and many other tissue-resident cells. ILCs provide an integrated view on how immune responses in tissues are synchronized with functional relevance far beyond the classical view of the role of the immune system in discrimination between self/non-self and host defense.

Codondex has evidenced p53 genetic variations, in multiple samples of same biopsy tissue from pancreatic tumors and oral squamous cell carcinoma's that may distinguish host tumor tissue gradients. The effect of highly-specific tissue-selected cell co-culture to educate ILC/NK cells may enhance the prospect for tissue penetration by these expanded, activated cytotoxic cells to improve overall survival.  

Expanding Treatment Horizons

An unrecognized link between p53 function and the immunosurveillance of cancer and infection led to an understanding how p53 influences the expression of MHC molecules at the cell surface via binding interaction with endoplasmic reticulum ERAP1.

Targeted mutations in multiple cancers revealed TP53 gene expression ranged between the 89th and 100th percentile of all expressed transcripts, and raised the possibility that p53 peptides arising from these common mutations might be immunogenic in these patients.

Select KIR-HLA composition favoring antitumor activity could be a promising immunotherapeutic strategy against breast cancer using autologous activated Natural Killer (NK) cell clones. Coexistence of inhibitory and activating killer-cell immunoglobulin-like receptors (KIR) to the same cognate HLA-C2 and HLA-Bw4 ligands conferred breast cancer risk. Inhibitory KIR(iKIR)-HLA pairs without their activating KIR (aKIR)-HLA counterparts were significantly higher in normal controls. Contrarily and adding complexity this suggests NK cells expressing iKIR, to cognate HLA-ligands in the absence of specific aKIR counterparts are instrumental in antitumor response. 

Identification and characterization of the peptides presented by HLA-C, G and E molecules has been lacking behind the more abundant HLA-A and HLA-B gene products. The peptide specificities of these HLA molecules were elucidated using a comprehensive analysis of naturally presented peptides. The 15 most frequently expressed HLA-C alleles as well as HLA-E*01:01 and HLA-G*01:01 were transfected into lymphoblastoid C1R B-cells expressing low endogenous HLA. 

The results (above) include allotype C*02:02 for p53 presentation and indicate the overlap of HLA source protein and top 500 peptides demonstrating the enormous complexity for multivariate analysis of immune response. However,  C*02:02 and C*05:01 have identical contact residues for p8 and p9, the residues of the bound peptide that influences HLA-C interaction with KIR. This suggests peptide effects could contribute to the broader and stronger binding reactions of these two HLA-C allotypes. Interestingly SART3 and MAGEA3 proteins both interact through the p53 pathway and are reported in the peptide study (above) in addition to TP53 to present ligands on C*02:02 and C*05:01. 

Moreover, in vitro  models demonstrated that p53 is required for upregulation of NK ligands. Further, there was a strong association between the KIR B haplotype and p53 alteration in Basal Cell Carcinoma (BCC), with a higher likelihood that KIR B carriers harbor abnormal p53 (p<0.004). Together the data suggests functional interactions between KIR and HLA modify risks of BCC and Squamous Cell Carcinoma and that KIR encoded by the B genes provide selective pressure for altered p53 in BCC tumors.

Notwithstanding the enormous complexity between iKIR, aKIR - HLA interactions, immunoterapy must address the highly specific characteristics of autologous precision and discover methods to sensitively educate NK cells so that minimally invasive treatments can be extended to patients who fall outside the patient cohort for strictly regulated treatments. 

Of course, its never that simple...

Chemo vs. Mecho

Data strongly suggests interaction between plasma membrane and submembrane at the endothelial surface controls the inflammatory response. 

A meta-analysis from six studies of global gene expression profiles of Blood Pressure (BP) and hypertension was performed in 7017 individuals. 34 genes were differentially expressed. Of these, 6 genes were linked including MYADM, which was the only gene, of 34 discovered across diastolic, systolic BP and hypertension. Knockdown of MYADM (19q13), a component of endothelial surface rafts induced an inflammatory phenotype altering barrier function through the increase of the adhesion receptor ICAM-1 (19p13). This is mediated by MYADM activation of ERM actin cytoskeleton proteins. 

Mechanical forces, without a definitive direction e.g., disturbed flow and relatively undirected stretch at branch points and other complex regions cause sustained molecular signaling of pro-inflammatory and proliferative pathways that include mechanical stretch tied to p53. 

ERM proteins also facilitate Sphingosine-1-phosphate (S1P) dependent egress for T-cells to migrate from lymphoid organs. Their directional migration, by blebbing is contained at the T-cell’s leading edge. This fundamentally different mode of migration is characterized by intracellular pressurization. Of the five S1P receptors S1P2 (19p13) is critical in the immune, nervous, metabolic, cardiovascular, musculoskeletal, and renal systems. Results suggest that the ratio between S1P1 and S1P2 (19p13) governs the migratory behavior of different T cell subsets. 

Human NK cells express S1P1 mRNA. Activation with IL-2 increases S1P1, promotes S1P4 (19p13) and S1P5 (19p13) but not S1P2 (19p13) expression. Unlike S1P1, S1P2 (19p13) signals through several different G-alpha subunits, Gi, G12/13, and Gq. S1P5 (19p13) is also expressed in human and mouse NK cells and was required for mobilization to inflamed organs. S1P5-deficient mice had aberrant NK cell homing during steady-state conditions. NK cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. 

Virus-infected mast cells selectively recruit NK cells and positively modulate their functions through mechanisms dependent on soluble mediators, such as interferons. Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1P2 (19p13) and releasing antimicrobial peptides. S1P2 (19p13),  a negative regulator of platelet derived growth factor (PDGF) induced migration and proliferation as well as SphK1 expression. 

S1P inhibits macropinocytosis (internalizing extracellular materials) and phosphorylation of Akt via S1P2 (19p13) stimulation resulting in diminished antigen capture.

S1P1, S1P2 (19p13) and S1P3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development. S1P2 (19p13)  and S1P3 are involved in regulation of endothelial barrier function, fibrosis, and vasoconstriction. 

Adipogenic differentiation is inhibited by S1P2 (19p13) as mediated by C/EBPα and PPARγ, which induces PEPCK, a more recent gene of interest in cancer that acts at the junction between glycolysis and the Krebs cycle.

Mecho or chemo, chicken or egg, what first?

Blood Pressure, Immunity and p53 Checkpoint.

Background

A few chromosome 19 curiosities developed into a deep-dive after looking into the primordial immune complex, the origins of MHC Class I and antigen receptors as revealed by comparative genomics. And the plot thickened because repressors (of endogenous retroviruses) that gained their binding affinity to retrovirus sequences at the same time their targets invaded the human lineage are preferentially located on chromosome 19. Further, the deletion rate in Zinc Finger clusters (ZNF) located around 19p.12 and 19q13.42, particularly between 51,012,739 and 55,620,741 are about twofold higher than the background deletion rate. A lot going on at this very active location which motivated this article.

At 19q13.42 kallikrein related peptidase (KLK’s), leukocyte immunoglobulin-like receptors (LILR’s) including killer-cell immunoglobulin-like receptor (KIR’s) as well MYADM, an important blood pressure related gene may also provide some clues to immunity variables that originate from or are influenced by this volatile region.

The retrotransposon bombardment of 19q13.42 and double background deletion rate is a significant remnant. However, after evolutionary MHC changed chromosomes ZNF, and within its range the chromosome 19 miRNA cluster (C19MC - 53,671,968 and 54,264,387) were still subjected to the deleterious effect of transposons. Regardless, suppression mechanics have kept epigenetic, regulatory and transcription processes, across gene’s far and wide on the move at a relatively stable rates. For example, reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues, but the effects of suppression may be sufficient to illicit a more permanent natural defense. In any event insertions and DNA damage are closely related and associated with loss of p53 that results in centrosome amplification. 

As cells pass through epithelial to mesenchymal transition (EMT), DNA damage prevents the normal reduction of p53 levels diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage.

Reproduction, Blood Pressure and NK

In reproduction, some of the 59 known miRNAs from primate-specific C19MC are highly expressed in human placentas and in the serum of pregnant women. They are also packaged into extracellular vesicles of diverse sizes, including exosomes and endow non-trophoblast cells with resistance to a variety of viruses. At least miR-517a-3p (a C19MC from fetal placenta) was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. miRNA's regulate the migration of human trophoblasts and suppress EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype. 

Maternal uterine or decidual Natural Killer cells (dNK) express AT1, AT2, ANP, proteins of Renin Angiotensin System (RAS) suggesting dNK have the potential to contribute to changes in blood pressure that occur between days 5 and 12 of pregnancy in mice. And, pressure related mechanical stretch on endothelial cells interconnects innate and adaptive immune response in hypertension.

Pressure variables in cells and tissues may result from infection, inflammation and membrane stretch, including inner mitochondrial membrane that affects electron transport chain, endoplasmic reticulum, antigen production, presentation and exosome bound p53 / miRNA release.  ANP colocalization to dNK’s suggests that dNK RAS, at day 12 infers a localized RAS related responsiveness. STAT3 in monocytes was activated by increased endothelial stretch and is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. The crosstalk between STAT3 and p53/RAS signaling controls cancer cell metastasis and cisplatin resistance via the Slug/MAPK/PI3K/AKT-mediated regulation of EMT and autophagy.

Educating NK Subsets 

Looking into some of the ~15 genes scattered among C19MC (~sixty miRNA's) between 53,671,968 and 54,264,387;

1. MYADM was one of two blood pressure signature genes (copper uptake protein the other) differentially expressed for systolic, diastolic blood pressure and hypertension. Of the ~35 identified genes, several more strongly related to immune cell functions including PRF1, GNLY, TAGAP, IL2RB, GZMB and CD97, NKG7, CLC that are located on chromosome 19. The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. MYADM controls endothelial barrier function through ezrin, radixin, and moesin dependent regulation of ICAM-1 expression an essential receptor for NK interaction.

2. PRPF31 is recruited to introns following the attachment of U4 and U6 (spliceosome) RNA’s. Experiments using PRPF31 determined p53 activation is a general consequence of interfering with the spliceosome. 

3. At 54,617,158 LILRB1 receptor is expressed on immune cells where it binds to 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 also educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

Monocyte/macrophage immunoglobulin-like receptors (MIR) genes are closely linked to the KIR gene family and the gene for FcαR at 19q13.4. The linkage was discovered in 1997 when a mouse sequence related to MIR mapped to a region on chromosome 7 syntenic with human 19q13.4. In 2012 a cluster of genetic loci, from multiple mouse strains and across anatomical sites was found to jointly contribute to the development of both thymic and splenic invariant natural killer T-cell NKT-cell levels. The dominant cluster was on mouse chromosome 7 and included almost all the non-C19MC genes located within the human C19MC region:– MYADM, CACNG7, VSTM1, TARM1, PRKCC(G), TFPT, NDUFA3, CNOT3, LENG1, TSEN34, RPS9. 

Four of nineteen knockout genes, that enhanced NK cell function were on chromosome 19 including GSK3 that phosphorylates Mdm2 to regulate p53 abundance, which would contribute to NK enhancement. 

A study of MHC disassortative mating in humans found Israeli’s were more gene similar, but MHC dissimilar than Europeans who were gene dissimilar and MHC dissimilar . Now, a recent study in American Indians found remarkably low KIR and HLA diversity in Amerindians that revealed signatures of strong purifying selection shaping the centromeric KIR region. This narrows to the importance of LILR-KIR region on chromosome19 that codes for the strongest NK cell educator receptors.

p53 regulates exosomes and miRNA’s directly influence NK responsiveness including regulation of dNK during pregnancy. Exosomes regulated by p53 also transfer it and can suppress growth and proliferation of p53 negative cells. Further, miRNA’s, induced by p53 can directly target ULBP2 mRNA and reduce its cell-surface expression.

Disease highlights

 

rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying miRNA-125b-induced apoptosis of lens epithelial cells. miRNA-125b is a novel negative regulator of p53. Deleting PRPF31 activates the p53 pathway and triggers retinal progenitor cells apoptosis. The members of the miR-125 family (miR-125a on chromosome 19q13.4 and miR-125b on chromosome 21q21.1) reside in two distinct human miRNA clusters with the let-7 and miR-99 families and these miRNAs are thus likely co-transcribed.

  

More succinctly, NK cells are alerted to induction of p53 in cancer cells by upregulation of the NKG2D ligands ULBP1 and ULBP2. p53 also induces expression of miR-34a and miR-34c, which target ULBP2 mRNA for destabilization. Observations suggest two possibly contrasting roles for p53 in NKG2DL expression and requires more investigation into how the regulation is fine-tuned. Extending this model to human populations would suggest that p53 must be inactivated among those with a robust NK response (those with B haplotypes). 

Taken together, our data suggest functional interactions between KIR and HLA modify risks of basal cell carcinoma (BCC) and squamous cell carcinoma, and that KIR encoded by the B genes provide selective pressure for altered p53 in BCC tumors. 

Conclusion

The convergence of several important cellular mechanisms that point back to a 19q13.42 address may illustrate ancient and conserved elements that perpetuate and function as integrated biological units effecting blood pressure, reproduction and immunity. Many of these impart education to innate immunity.

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. 

Systolic Blood Pressure and Innate Immunity vs. the Cancer Brain

Participants with a valid heart disease phenotype (atherosclerosis) were identified in a MESA blood pressure analysis conducted over 10 years. The valid group varied from 770 to 1113 patients from whom further blood analysis queried a primary and exploratory hypothesis of immune cell subsets. Four statistically significant innate cell subsets were discovered to be associated with Systolic blood pressure (SBP); Natural Killer (NK) cells, gamma delta T cells and classical monocytes.

Separately, an analysis of 7017 individuals from 6 international studies of gene expression signatures for SBP, diastolic blood pressure (DBP) and hypertension (HTN) found 7717 genes of which 34 were most differentialy expressed. Enrichment analysis for the systolic and diastolic gene group's associated to NK cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response, pointing strongly to innate and adaptive immunity. MYADM was the only gene identified for all groups SBP, DBP and HTN.

MYADM controls endothelial barrier function through ezrin, radixin, and moesin (ERM)-dependent regulation of ICAM-1 expression. ERM expression is required for ICAM-1 expression in response to MYADM suppression or TNF-α. ICAM-1 is a paradigmatic adhesion receptor that regulates leukocyte adhesion together with integrin LFA-1. This connection between endothelial membrane and cortical actin cytoskeleton appears to modulate the inflammatory response at the blood tissue barrier. 

Pressure overload activates the sympathetic nervous system (SNS) and up-regulates p53 expression in the cardiac endothelium and in bone marrow (BM) cells. Increased p53 expression promotes endothelial-leukocyte cell adhesion and initiates inflammation in cardiac tissue, which exacerbates systolic dysfunction. SNS activates, at least by significant increase of circulating norepinephrine (NE), which up-regulates p53 expressions, while forced expression of p53 increased ICAM-1 expression. 

On endothelial cells SNS is mediated via catecholamine-β2-adrenergic signaling, which up-regulates the production of reactive oxygen species (ROS), activates p53 and induces cellular senescence. Immune cells, including macrophages, monocytes, NK cells, B and T cells express the β2-adrenergic receptor and catecholamine. During pressure overload, NE cultured macrophages up-regulated p53 expression, whereas introduction of p53 increased Itgal (LFA-1) expression (which binds ICAM-1). Treatment with NE increased ROS, which was attenuated after inhibition of β2- adrenergic signaling in macrophages. Endothelial cell–macrophage interaction via NE-ROS-p53 signaling induces up-regulation of adhesion molecules, thus contributing to cardiac inflammation and systolic dysfunction.

During hypertension the vascular endothelium activates monocytes, in part through ROS by a loss of nitric oxide (NO) signaling, increased release of IL-6, hydrogen peroxide and a parallel increase in STAT activation in adjacent monocytes. NO inhibits formation of intermediate monocytes and STAT3 activation. Humans with hypertension have increased intermediate and non-classical monocytes and  intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3.

A senescence-associated secretory phenotype (SASP) was induced in epithelial cells after DNA damage of sufficient magnitude. In premalignant epithelial cells SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy by a paracrine mechanism that largely depended interleukin (IL)-6 and IL-8. Strikingly, loss of p53 and gain of oncogenic RAS exacerbated the pro-malignant activities. This suggests a cell-non-autonomous mechanism by which p53 can restrain and oncogenic RAS can promote the development of age-related cancer by altering the tissue microenvironment. Oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. 

Induced stretch or stretch from pressure overload may engage a non-autonomous, p53 centric micro-mechanical mechanism that escalates or deescalates innate responses against cells functioning outside the mechanical ranges that macrophages or NK cells permit. Thus, the neuro-immune extension through SNS signaling, may begin with circulating blood pressure or stretch promoted through inflammation. 

Genetic Eruption and p53 Response!

L1 are a class of transposable DNA elements found in 17% of the genome that are evolutionarily associated with primitive viral origins. Around 100 have retained the ability to retrotranspose. Without restraint they can interrupt the genome through insertions, deletions, rearrangements, and copy number variations. L1 activity has contributed to instability and evolution of genomes, and is tightly regulated by DNA methylation, histone modifications, and piRNA. They can further impact genome variation by mispairing and unequal crossing-over during meiosis due to its repetitive DNA sequences. Indeed, meiotic double-strand breaks are the proximal trigger for retrotransposon eruptions as highlighted in animals lacking p53.

189 gastrointestinal cancer patients across three cancer types: 95 stomach, colorectal esophageal were examined for any aberration in DNA repair pathways that could be associated with L1 retro-transposition. Out of 15 DNA repair pathways, only the TP53 repair pathway showed a significant association. L1 retro-transposition is inversely correlated with expression of immunologic response genes including interferons. Frequent TP53 mutations in tumors with a higher load of L1 insertions suggest the critical role of TP53 in restricting retrotransposons as a guardian of L1 expression and cancer immunity.

A screen of 172 open reading frames (orfs), of unknown genetic function across several human viruses was designed to discover novel interactions with p53. The orfs encoded viral proteins, miRNA's and lncRNA's. The ORFEOME project was based on the hypothesis that every virus should encode some functions that interfere with the p53 signaling network. The methods present a broad net by screening for interactions without necessarily defining how interactions arise.

The DNA damage response (DDR) pathway stabilizes p53 leading to increased nuclear relocation, binding to p53 response elements, rearrangement of chromatin and transcription of p53 target genes. Any of the multiple p53 related interactions along the way is a potential target of translated viral proteins on the function of p53. 

p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling by the induction of genes containing IFN-stimulated response elements. p53 also contributed to an increase in IFN release from infected cells. This p53-dependent enhancement of IFN signaling is dependent to a great extent on p53 activation and transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Thus p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

p53 likely cooperates with histone and DNA methylation to silence specific retroelements. In the zebrafish model, it was shown that p53-dependent H3K9me3 methylation, in the promoter region of a synthetic human LINE1 element mapped to a known p53-binding site. Some evidence in human cell lines suggests that p53 can physically interact with both H3K9 tri-methyltransferases and DNA methyltransferases. In basal stress-free conditions, unacetylated p53 is pre-bound to many target genes together with SET - a repressor protein, which mediates repression of p53 target genes. Additionally, p53 as a master regulator of transcription might regulate gene expression of key epigenetic or piRNA factors. 

Through L1's we get a sense of p53's interconnectedness to DNA damage, viral replication, cancer and immunity. In a way we can sympathize with it, especially when overloaded by viral infiltration and eruption. Its understandable how, under those conditions double stranded DNA breaks and pathway impediments compromise its ability to be guardian of the genome!

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.

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.