Natural Killer cells and Innate Immunity

Killers not as natural as they appear

Within the ecosystem that is our body, Natural Killer (NK) cells are highly selective predators: in a few hours they kill infected cells or tumors while sparing healthy cells.

Throughout the world, scientists have combined their efforts to understand how these killers eliminated only these targets, even though they lacked the highly selective antigen receptor used by their close cousins, the T cells

Today we still need to know better how NK cells are controlled and how they in turn control other agents of the immune system. The history of NK cells is thus far from over. Upon entering into the lair of the predator, Eric Vivier, Sophie Ugolini and the team have contributed to discover that these natural killers do not seem to kill as naturally as we thought.

N for Natural, K for Killer, as its name suggests, NK cells are primarily killer cells. With their congeners in the innate immune system (neutrophils, monocytes, macrophages, dendritic cells and Tγd lymphocytes) they patrol the body (they are found in the blood but also in the liver, spleen, lung, intestine and lymph nodes) and identify cancer cells or cells infected by a microorganism. Once identified, the diseased cell is destroyed in minutes by a mechanism known as cytotoxicity: the NK cell attack in a melee by releasing substances that perforate the "skin" of their victims. Death is by cell lysis.

NK cell (left) iinteracting with a tumor cell (right). Courtesy of Christina Tramba, Cancer Council of Tasmania

Meanwhile, the NK cells secrete cytokines (the "hormones" of the immune system) that stimulate and guide the response of other agents of immunity such as macrophages, dendritic cells or lymphocytes of the adaptive immune system. Using their highly selective receptors (antibodies attached to the surface of B cells and T cell receptors), T and B lymphocytes identify antigens that mark the taget cells.
Lacking such key recognition elements, how do NK cells manage to distinguish a diseased cell from a healthy cell? The lab was instrumental in solving this puzzle in the mid 90s.

"During evolution, the NK cell just learned to count! "

"To distinguish the normal from the pathological, the NK cells have developed a highly sophisticated detection system," said Eric Vivier. "This is based on "surface radars", coupled to intracellular signaling pathways that transmit information to the cell so that it reaches its decision: kill or not kill. Among these radars, there are activating and inhibitory receptors. The first spots the danger signals emitted by stressed cells and put the NK cell in an "extermination" mode. The second detect self molecules (such as the Major Histocompatibility Complex class I or MHC I molecules) expressed on all the healthy cells of each individual. These deactivate the effector function of NK cells: the target is spared and the predator can then get back on patrol;"

However, most of the time, the NK cells receive these activating and inhibitory signals simultaneously. In this context how do they manage to make a decision?

"In the course of evolution, they just learned to count! " said Eric Vivier. "The NK cell 'sum's the signals it receives: it spares healthy cells which send inhibitory signals and little or no activating signals, while it kills cancer cells or infected cells that not only deliver ‘danger signals’", but also become unable to send protective inhibitory signals. In sum, the NK cell functions like an ideal drug: effective and safe."

Looking through the succession of the handful of molecular signals that lead from the surface to the cell nucleus, the team made another surprising discovery: in the vicinity of the transmembrane receptor activators they found a small protein (named KARAP/DAP12/Tyrobp) which carries a signaling motif, called ITAM (immunoreceptor tyrosine-based activation motif), without which the activation no longer occurs. At the same time, they revealed the existence of a motif called ITIM (immunoreceptor tyrosine-based inhibition motif) in the inhibitory receptors (KIR in humans, Ly49 in mice and CD94/NKG2A in both species). Like its activating counterpart, the ITIM conditions the responses of the NK cells, in this case, stopping the mechanisms of activation.
Found on many cell types, these motifs are now considered "universal biological switches" through which signaling activity is controlled.

The identification of molecules recognized by activating receptors is one of the new priorities of the team. Indeed, if the ligands for KIR inhibitory receptors are known (MHC I molecules of the self), those of the activating receptors remain to be discovered. "For this we generated antibodies against tumor cells expressing the molecules that interest us," says Sophie Ugolini, co-head of the team. "The goal is then to select antibodies that are able to block the cytotoxicity of NK cells to isolate the corresponding ligand."

Innate immunity not so innate as it seems

In parallel, the team attempts to disassemble the mechanisms that regulate the operation of NK cells. Unlike the classical approach to inactivate a previously identified gene to characterize its function (reverse genetics), they have tried another approach, which is to try to find the genes that respond to the question (forward genetics).
"To identify, for example, genes involved in the recognition of tumor cells, we induce random mutations in the mouse genome using a chemical agent: ENU," says Sophie Ugolini. "We cross our mutant mice together to obtain a strain carrying a mutation of interest for the biology of NK cells, then we go back to the mutated gene using new generation whole genome resequencing strategies."

Through this systemic and unbiased approach, the team has pinpointed a number of genes involved in each step that lead to the removal of the tumor: the activation of certain genes, the synthesis of receptors, the lytic process ... but also the learning process of the NK cell!

Why would "natural killers" supposed to kill spontaneously, not need prior training? "Just because this concept is wrong and the word natural totally inappropriate" reveals Eric Vivier. "The function of NK cells is by no means ‘natural’; like the killer T cells of adaptive immunity, NK cells first need to be initiated in order to kill. "

Surprisingly, the team contributed in showing that prior learning was involving inhibitory receptors. Thus, if NK cells are not stimulated through their inhibitory receptors, they remain hyporesponsive. "With the nano-imaging methods developed by the team of Didier Marguet and Hai Tao He, we found that this commitment leads to a dramatic reorganization of activating receptors on the surface of the NK cell and demonstrated that this rearrangement makes NK cells much more effective killers," concluded Eric Vivier.

A new therapeutic option?

Thus, by killing their targets selectively and in communication with their environment, natural killer cells protect us from pathogens (herpes, cytomegalovirus...) and amplify or limit (in the case of autoimmune diseases) immune responses. Yet their role in vivo is still debated.

The team is now trying to better understand their role. "The ideal would be to have a mouse model in which we can selectively remove NK cells "ad libitum". That's what we've done" reveals Eric Vivier. "It remains now to confront it with viruses, bacteria and tumors to see how this mouse mutant is able to respond to these various attacks."

In reality, our NK cells are not always able to effectively combat pathogens and cancers we face. Hence the idea of building on these discoveries to boost the effectiveness of NK cells. For that, two approaches are possible a priori: increase enhancing signal strength by mimicking their receptor ligand activators or abolish all or part of the inhibitory message by blocking their inhibitory receptors using an antibody. This second approach was chosen by Innate Pharma, a company created in 1999 in part from the work of Alessandro Moretta, Professor at the University of Genoa, and Eric Vivier.

This drug is currently being evaluated in two clinical blood cancers with poor prognosis: acute myeloid leukemia and multiple myeloma. Beyond their therapeutic value, these tests may reveal many other facets of the biology of NK cells.