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.  

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.