The most fundamental characteristic of life is replication. The minimum unit of life, a cell replicates itself by division. The genetic material, DNA, is duplicated, which encodes the blueprint of an organism. In the case of prokaryotes like bacteria, one copy is kept in a parent, and the other is delivered into a newly formed daughter cell. In the case of eukaryotes, such as multicellular organisms like us, a parent cell is separated into two daughter cells so that each inherits the exact copy from the parent. In either case, one cell becomes two with maintaining the original property. This is a successful replication. Life goes on. If this mechanism is 100% accurate, the original is perfectly replicated each time. No change. No evolution. If it is too loose, there is no consistency and continuity. In real life, we are at a narrow tipping point. Not too tight and not too loose. The level of integrity and looseness is just right, as humans have evolved 3.7 billion years since the first life emerged.
An essential thing for the integrity of replication is recognizing what to replicate—in other words, recognizing SELF and non-SELF. In analogy, this is like recognizing how you separate your property from someone else’s. For example, when I ask my daughter to clean up our common living room at home, she only does it for her stuff but leaves others. No energy is spent on others—the same idea. Even a bacterium needs to know which DNA sequence is it's own. Otherwise, someone else may piggyback. How does it do that? Can it put name tags on its DNA sequence?
Bacteria use two strategies to distinguish SELF and non-SELF in DNA sequences. We can say this as defence (immune) mechanisms maintaining the SELF-integrity of genetic information by excluding non-SELF. Both strategies recognize non-SELF. Thus, something not recognized as non-SELF will become SELF. One of the two systems is restriction enzymes, sequence-specific DNA endonucleases. The double-strand DNA carrying enzyme-recognition sites will be cut and digested. This means the DNA sequence getting cut by its restriction enzyme is recognized as non-SELF. To make this strategy work, one’s DNA sequence must not have the recognition sites. It is simple but effective. We use the same concept when we take synthetic fake photos (i.e. a green background can be removed and replaced with something else). The disadvantage of this system is limiting their genome size. If an enzyme recognizes a four-base-pair motif, called a four-base-pair cutter, the chance to have it in a random DNA sequence will be 1 in 256. A six-base-pair cutter will theoretically cut every 1 in 4096. The four-base-pair cutter is sensitive to detect non-SELF, but the risk of having the enzyme recognition sequence accidentally due to replication errors is higher than the six-base-pair cutter. This suggests that this strategy limits genome size to the fidelity of DNA replication.
The other strategy is the CRISPR/Cas system. This system is adaptable. The primary method to detect non-SELF sequences is the presence of the PAM sequence. The PAM sequence is usually very short and specific to each species. If there is a PAM sequence, this DNA sequence has the potential to be non-SELF, while if there is no PAM, the DNA sequence is SELF. Do all DNA sequences carrying PAM get cut? No. If the cell itself or its ancestor had been challenged previously by the same DNA sequence, like a bacterial phage (a virus infecting bacteria is called a phage), pieces of the viral sequence adjacent to the PAM motifs in the phage will be incorporated into own bacterial genome at the CRISPR allele. Then, the next time the same bacterial phage infects a bacterium of the progeny of the original infected bacterium, it activates the CRISPR/Cas system. Because its CRISPR allele has identical sequences of the phage genome, when transcribed and processed to form Cas9 RNA-Protein complex endonucleases, they primarily recognize the PAM motif and then recognize the target sequence. If the sequence is matched, the double-strand DNA will be cut in a sequence-specific manner. Therefore, this system has genetically inheritable memory against specific DNA sequences. Non-SELF is recognized in restriction enzyme and CRISPR/Cas systems, and SELF is the default.
For multicellular organisms, the importance of integrity and continuity shifts to the organismal level from the cellular level. This permits larger genome sizes in eukaryotes. If a pathogen like bacteria (prokaryotes) or virus invades, the organism must recognize it. The primary cell population responsible for this is ameba-like phagocytic cells, macrophages. They recognize a pathogen and eat it up. How do they recognize it? Using Pathogen Pattern Recognition (PPR) receptors, represented by toll-like receptors (TLRs), recognizing unique patterns in bacteria and viruses. Bacteria and viruses have unique molecular properties we, eukaryotes, do not have. Once ancient metazoans developed the receptor system to recognize the properties common in bacteria and viruses, all descendants inherited it. This is called innate immunity.
Interestingly, this innate immune system, primarily phagocytic macrophages, also recognizes its cellular damage. If a neighbour cell is damaged, macrophages can recognize it for clearance. How do they do that? A cell is a sac that consists of a lipid bilayer called a plasma membrane. Various unique biological materials like ATP, histones, and mitochondrial DNA are packed within a cell. Those precious biological materials are only present inside a cell but absent in the external environment. When plasma membrane integrity fails, like by osmolality shocks, those molecules are leaked into the extracellular environment. Then, they are recognized as Damage Associated Molecular Patterns (DAMPs) by innate immune cells using the PRRs and other receptors. Both PPRs and DAMPs are evolutionary consistent as molecules. Thus, their receptors are also conserved and selected as an advantageous trait. The main cellular player to detect pathogens and cellular damage is macrophages. Some macrophages work like residential police guards distributing all internal and external surface areas of the body, where the pathogens initially invade. Others work like cruising police cars circulating the bloodstream. Both sense pre-determined specific patterns to detect pathogen insults and cellular damages.
The adaptive immune system, like B-cells and T-cells, is unique in vertebrates. This is the only system recognizing SELF. Except for SELF, all others will be potentially recognized as non-SELF. Making this conceptually reverse condition work is more complex than you think. You need to know all SELF. The previous three systems that define non-SELF establish a relatively clear boundary between SELF and non-SELF. The default is SELF. But in this defining-SELF system, the border needs to get blurred; back to my daughter’s story. Her properties can be definitively hers, sometimes her brother’s, often the family’s are hers, proudly her school’s and even her country’s are hers (i.e. Japan and Canada for her). In addition, when she purchases something new, it will become hers from others. Hers can be context-dependent and changeable with time.
Similarly, if an organism tries to identify SELF, it will be less sharp boundary than defining non-SELF. How can this be achieved? The combination of the abundance of SELF-antigens and the affinity of its receptors sets the boundary between SELF and non-SELF.
Training is essential for this strategy because the organism does not intrinsically implement the boundary between SELF and non-SELF. For example, T-cells recognize unique antigens with its receptor, called T-cell Receptors (TCRs). Each T-cell has one unique receptor type on its surface. T-cells can kill the other cells in the body but cannot directly kill bacteria or eliminate viruses. For T-cells to activate their killing activity, the antigen needs to be presented by other cells. In my analogy, any modern customer does not eat a chicken on the street but the ones sold in a supermarket. They are nicely processed and packed on a tray. Then, we will buy and eat it—the same idea. T-cells only deal with the antigens properly processed and packed on a tray in each cell. They do not directly recognize anything in the extracellular space unless processed, packed and presented by a cell.
The mechanisms by which their receptor variation (TCR repertoires) is established have been studied and reasonably well understood. This happens within a small organ in our chest, the thymus. Specific cell populations in the thymus train and select T-cells. First, the T-cells that properly recognize the tray are chosen to survive (positive selection). Then, the T-cells that recognize the epitopes derived from own SELF-proteins are killed (negative selection). The only ones that survive are T-cells that can recognize the tray but not recognize SELF-proteins. The molecular trays presenting an epitope on the cellular surface are called MHCs. All people have two types of trays, MHCI and MHCII, but each person has distinct subtypes inherited from their parents. All somatic cells in the body express MHCI. Antigen-presenting cells (APCs) such as macrophages and dendritic cells have a higher phagocytic activity and express MHCII, in addition to MHCI.
In normal somatic cells, each cell type only expresses its specific functional proteins, like neural proteins in neurons and muscle proteins in muscles. Interestingly, the thymic cells can express various proteins to display them on MHCs only for T-cell training. Therefore, many kinds of proteins specifically expressed in unique cell populations are also presented in the thymus. Then, any T-cells carrying TCRs strongly reacting to own protein epitopes will be eliminated (negative selection). On the other hand, T-cells weakly recognizing them derived from SELF-proteins will be released into circulation. They are controlled by another layer of regulation, peripheral tolerance. The precise control of peripheral tolerance is significant because eliminating all autoreactive TCRs, including weak ones, creates a high risk of making a gap in the TCR repertoire.
Importantly, the affinity of TCRs against peptides alone does not set the boundary between SELF and non-SELF. Not that simple. A combination of the affinity of TCRs and the abundance of peptides within each cell determines it. In analogy, the MHC repertoire on a cell is a show window of a small stop. Each shop has a limited display space. Not all products will be displayed on the shelves at once, and something sold more often takes more space. Competition. One abundant peptide (e.g. a top-rated product in a shop) would take up one full top shelf all the time. On the other hand, low-abundant peptides would have a chance to have one tray on one bottom shelf or even just one day per week. In a normal homeostatic condition, the MHC repertoire displays its cellular identity. Regular customers know which shop sells what items based on its display.
Within weakly SELF-reactive T-cells, the abundance of their targeting peptides varies in each cell. TCRs against low-abundant peptides (i.e. only displayed a tiny portion of the MHC repertoire at low frequency) have no issue in normal conditions. On the other hand, TCRs against high-abundant peptides in specific populations need to have another regulation: peripheral tolerance. Thus, the abundance of a peptide within each cell and the TCR affinity against the peptide will set the boundary of SELF and non-SELF.
The unique power that T-cells possess is killing own cells. If a somatic cell displays the peptide that a TCR can recognize on a tray with a surge of inflammation, the T-cell can kill this cell. This could happen in the situation of viral infection. When a virus infects a cell, the cell senses viral infection based on innate immune reactions to detect cytoplasmic DNA and dsRNA and activates type1 IFN inflammatory response. The virus replicates within the cell and eventually lyses it to come out to infect thousands of surrounding cells. During this process, viral-derived epitopes will occupy the MHC repertoire of the cell. In analogy, the show window of your favourite shops suddenly changes to something you dislike. Any T-cell recognizing viral epitopes can kill the cell before viral-inducing lysis. But probably, the first-time infection would not be efficiently removed because the functional avidity of naïve T-cells is not high. Unlike BCRs in B-cells, T-cells do not mature the TCR affinity against antigens but enhance TCR responsiveness. Previous immunization (vaccination) is a matter of preparation for T-cells with a high functional avidity against viral epitopes, thus can induce a quick killing response.
Interestingly, neither recognition by TCR alone nor inflammation alone is sufficient to kill the cell. Only the simultaneous two stimulations can activate the killing activity of T-cells. Thus, there is no killing if a naïve T-cell recognizes its antigen on a cell without inflammation. The antigen will be trained as SELF and establish local immune tolerance in this case. A naïve T-cell carrying this TCR will differentiate into a regulatory T-cell. Importantly, this is the process of normal homeostasis. Without this peripheral tolerance, an animal develops autoimmune conditions. Whether or not the antigens are derived from their genome is not essential at the peripheral level. Whether inflammation is accompanied or not at the first encounter of an antigen and its TCR defines the SELF/non-SELF boundary of the rest of life.
Building the SELF/non-SELF boundary is essential for the integrity and continuum of species. Defining non-SELF is relatively easy and permits a sharp clear boundary. From procaryotes to eukaryotes/metazoans, the recognition mechanisms change from the genetic sequence to the organismal property. Although metazoans do not use sequence-based recognition, innate immunity that all metazoans have recognizes common properties of pathogens, non-SELF, and cellular damage. When vertebrates evolve, adaptive immunity is established. The acquisition of adaptive immunity allows the vertebrates to have relative longevity and larger body sizes. As a trade-off, we gain unique diseases like cancer.
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