Ex Vivo culturing of NK cells and infusion

I guess this is a transition from my recent CAR obsession. NK (natural killer) cells are the other killer lymphocytes than Killer T-cells. They don't have an antigen specific receptor. Instead they have antigen non specific activation receptors (including NKG2C and NKG2D) and, importantly, Fc receptors which bind antibodies then kill the cell to which the antibody sticks (antibody specific cellular toxicity). This means that there is a flexible antigen specific response to any antigen which induces an antibody. That means that they are like natural CAR T-cells too in a way, but with no genetic modification. It is almost odd that growing NK cells outside the patient and infusing them is a fairly small effort reported in a fairly small literature which (as always) I will not responsibly cite (feininger, Mortier, Felicias, Miller sp???). One issue is that the NK cells do not multiply when activated. I think this is not a big problem as they can be stimulated with IL-15 (this requires continuous infusion which is not such a huge hassle). Another problem is that grown for a long time ex vivo with lots of IL15 and also IL12 (too inflamatory to put in patients) and (I think) IL20, they become hyporesponsive, anergic, inactive. (TL:DR version -- I think that it should be checked whether this problem is prevented if the NK cells are grown along with activated macrophages -- the post is definitely TL). There is an article on this (no cite) which says the problem can be solved by one week stimulate with interluekins, then one week pause, then etc. The article also asserts that the problem is that prolonged MTOR activation causes cells to turn off the system for fatty acid oxidation which means that they literally don't have the energy (as in calories) to do their job. I have to mention this, because I want to present a competing hypothesis. I think that NK cells become hyporeactive if they have been too long since touching an activated macrophage. Here I really should cite (all articles include as an author TA (thomas) Waldmann, my late father). This includes "preconditioning ..." Sato, Banfield Tagaya, Waldmann) which notes that naive cells do not respond to IL15 until they have touched a macrophage. "FC IV" Zhang, Anton, Waldmann, al et Ravetch on how NK cells exposed to IL15 and macrophages express an FC receptor which irreversibly binds antibodies. trans endocytosis Anton Waldmann and others The story is that when lymphocytes are in contact with activated macrophages, they engulf little bits of the macrophage forming vescicles with 2 layers with IL15 on the outside of the inner membrane and IL15 beta-gamma receptor on the inside of the outer membrane, so they constantly have IL15 stimulation. This is mainly MTOR not Jak-Stat stimulation (won't define but I think important). If the NK cells divide in pure NK cell culture, they dilute the vescicles which don't divide. I think this may be the problem. In any case (in case anyone has read this far) I think the solution is to add activated macrophages to the solution. Culture a mixture of NK cells and macrophages with IL15, IL12 (and I think IL20). One thing is that, like NK cells, macrophages are ADCC (antibody dependent cellular toxicity) effectors. Last thing (this is due to dad) is that it is important to activate the macrophages with anti-cd40 or Toll like receptor agonists such as lipopolysaccaride (endotoxin) RNA, poly GC DNA (with unmethylated C) c-reactive protein or well there are lots of otpions and a large literature. This is a very simple experiment. I think it is worth doing.