Immunology for Experts!

If you really want to know more about immunology, this section is for you! The information provided in the links come mostly from the remarkable immunology course University of South Carolina, School of Medicine (congratulations to Richard Hunt and his team for this wonderful contribution!).

As an introduction, you will find a  video recapitulating some of the critical steps of our immune response.  It recalls that the immune response takes place in two stages: an immediate response after microbe infection called " Innate or Natural Immunity" provided by dedicated cells followed by a delayed immune response (delayed by a few days) called "Adaptive or Acquired Immunity" provided by specialzied cells, the lymphocytes. The molecular determinants  found on pathogens and triggering the reaction of our body are markedly different for these two kinds of responses!

Video provided by GarlandSciences

Let's first introduce the actors (cells and organs) of our immune system.
Some immune cells
  Most cells of our immune system, flowing in the blood, were discovered long ago, mainly by the Nobel Prize winners Paul Ehrlich and Ilya Metchnikov Some of them patiently wait in our tissues for microbe crossing the boundaries of our body (skin,  lung or intestinal epithelia or blood. Then these cells react against the pathogen. These cells are kind of border guards called Mast cells, Macrophages or Dendritic cells Upon entry of a microbe, these cells recognize it and produce weapons to fight against it. To avoid being overwhelmed, these cells create the conditions for more immune cells to reach the infected tissue: cells that circulate quietly in our blood can quickly intervene if necessary and will then leave the blood to reach infected tissue and fight against the microbe (this is called diapedesis). Among these cells, one can find Neutrophils, Eosinophils or Basophils that belong to the granulocyte population. This term refer to the rich content of these cells in intracellular granules, which store molecules active in immune response). The granule content can be released through the degranulation process to gain access to the pathogens. Granulocytes, and, in particular, neutrophils are the fastest cells to exit the blood stream to reach the infected tissue, prompty followed by Monocytes, and bothe act together to kille the microbes (this occurs within a few minutes after infecetion). Other cells give an air of "polar" to the immune response: these are NK cells or Natural Killer which, as their name suggests, are killer cells. They actually kill our own cells when they are infected by a microbe or become cancerous
NK cell (yellow) attacking a tumor cell

Dendritic cells are cautious: at the entrance of the microbe, they leave the infected site and will move to specialized organs called secondary lymphoid organs, to alert potent cells of our immune system: the T  and B Lymphocytes. These cells, once informed of the presence and identity of the microbe by dendritic cells,will take the time to equip themselves with the most suitable weapons to fight against the microbe in question. Then, we will leave the seondary lymphoid organ to move back to the site of infection and eliminate the microbe (when all goes well!). It takes a week before all this happens. These cells are also very important because they are the "immune memory" ie if the same bug again crosses our borders, these cells will remember the best way to eliminate it and make it in a much shorter time than for the first infection.
To become more familiar with the appearence of all these cells, you can watch a remarkable video describing the blood cells!

To test yourself on the immune cells, nothing but a quizz!(sorry it is currently in french...)

White blood cells analyzed by flow cytometry
To analyze both qualitatively and quantitatively the cells of our immune system, one of the most effective techniques is flow cytometry. It is a technique that uses a very sophisticated equipment that enables a detailed characterization and identification of cells that look very similar otherwise. Flow cytometry can also be used to understand how these cells fight against microbes. It is based on the phenomenon of fluorescence emission, which vares with the size and content of cells. In addition, flow cytometry makes use of molecular tools, antibodies (yes, the same produced by our immune system) to which a fluorescent molecule can be attached. Antibodies have the ability to lock onto a target molecule and to recognize in selective manner cells displaying the moecular target. Like "missiles", these antibodies will be guided to the cells, will locking onto them and send the light signal which allow their identification by the cytometer. This technique has revolutionized knowledge in immunology! In general, many techniques in biology are based on the  reaction between antibodies and antigens.

Lymphoid organs
Lymphoid organs are specialized organs of our body where the cells of our immune system develop, grow, and are recruited into an immune response against an infectious agent. It is in our bone marrow (yum!) that most cells of the immune system are produced but some of them (the famous T lymphocytes) finish up their development in the thymus (yum!). T cells are themselves a heterogeneous population of cells: the most famous ones are the CD4 and CD8 T cells. It is in the thymus that T cells choose to become one or the other of these sub-families each of them having specific roles in immunity. These organs are called primary lymphoid organs as they serve the production of cells. The organs where lymphocytes are recruited in an immune response and armed to fight against pathogens are of a different nature and are called secondary lymphoid organs are (those, do not eat...). This is for example the spleen, tonsils, appendix, lymph nodes and Peyer paches.  In these organs, a series of very well coordinated and orchestrated events occur. Circulating lymphocytes pass by these organs on a regular basis desperingly looking for..antigens! Again, you can watch a remarkable histological analysis of the  bone marrow, of the thymus and of the lymph nodes.

T and B cells at their right place!
Throughout their life, cells of our immune system either move on short distances, on long distance or remain immobile in tissues.  All these movements and positioning are tightly controlled  during the immune response. They result from the combination of adhesion phenomena (achieved through a kind of molecular velcro present in our cells) and migration phenomena made under the influence of substances attractive to our cells (this is called chemotaxis).

This lovely film shows the movements of an immune cell under the influence of a "chemotactic" stimulus ie by a chemical molecule called "chemokine" that triggers cell migration. You will be able to measure the real-time reactions to the displacement of the cell toward the source of the stimulus. In fact, the cell "search" for the stimulus source (ie it is directed towards areas where the stimulus is the most concentrated): it works the same thing in our body!
Another film shows how the chemotactic
receptors work in response to these stimuli.

Let's see now how our innate immune system recognize and fight against germs .
A Toll-Like Receptor
The receptors found on cells of the immune system capable of recognizing the microbes belong to a family of receptors called PRR (stand for Pattern Recognition Receptor). These receptors found on cells of the innate immunity recognize molecules commonly found on large classes of microbes but absent from our own cells. PRR recognize certain molecules present on bacteria or viruses for example. Wanna travel through a PRR structure? Take a seat and ravel within the structure of one of these receptors, the TLR (Toll-Like Receptor). These receptors are often subtle enough to distinguish between two main families of bacteria. Once these receptors detect the microbe, the cell bearing the receptor is activated. Sometimes this allows the cell to literally "eat" the microbe (eg during the phagocytosis process illustrated by a movie), sometimes it allows the cell to produce anti-microbial weapons or chemical molecules that enable communication between the cells of our system immune. In the case of dendritic cells, PRR stimulation results in a radical change in the biology of the cell. Within a few hours, the cell replaces its ability to detect microbes by the ability to activate T lymphocytes This is accompanied by morphological changes and by the migration of these cells from the infected tissue to the neares secondary lymphoid organ. NK cells play a somewhat particular role in the innate immune system as they take care of infected cells and tumor cells rather than directly killing microbes. How these cells decide to kill or leave our cells alive is a complex process. NK cells choose killing our cells when they detect stress molecules on the surface on these cells or, conversely, when certain molecules, namely HLA class I molecule disappear from the surface of our cells. These events occur when our cells. HLA disappearance often occurs when our cells are infected by viruses for example, or when they are cancerous.

Inflammatory reaction
Recognition of microbes by innate immune cells often triggers the inflammatory response. Inflammation is a constant in the immune response. It is manifested by redness, heat, swelling and pain in the infected tissue. It is sometimes accompanied by a general increase in heat (fever). You've all experienced these symptoms, sometimes only giving you a shot. Indeed, inflammation is quite similar after an infectious or a traumatic cause! Many cells and molecules (such as lipids, amines, cytokines, neuropeptides) are involved in its onset, its maintenance and its resolution. Indeed, inflammation must be tightly regulated in time and intensity. Otherwise, you will suffer from inflammatory pathology and take anti-inflammatory drugs (aspirin is the best example as paracetamol, ibuprofen, cortisone etc. ...). You will enjoy to see a movie that will explain how a receptor for molecules involved in inflammation such as cytokines works.

The complement system: initation of the cascade and function
--> Ultimately, these reactions allow cells of our innate immunity to destroy microbes or infected cells. The innate immunity makes use of various strategies for getting rid of microbes or infected or cancerous cells. These include the action of phagocytes, killer cells such as NK cells and also complement. Phagocytes perform phagocytosis ie, after recognizing a pathogen, these cells swallow and destroy it literally by putting it in contact with very harmful substances (pretty much like the gastric juices digest the food we have ingested). NK cells destroy infected cells and tumor by sticking to them and pouring molecules which perforate (perforin) the membrane of the target cell, leaving the passage for molecules (granzymes) which will enter the cytoplasm of target cell to induce its destruction (see movie). Eosinophils use a similar strategy (although with different effector molecules) to destroy parasites. Finally, the complement system can, among other things, opsonise the bacteria and sensitize it to improved destrcution by phagocytes or directly kill it through the mobilization of a membrane attack complex, which perforates the bacterial cell wall (see animation).

--> Let's now see how to mobilize our adaptive immune response, and in particular, how are pathogens recognized and eliminated by this immune response.  --> As you already know, an adaptive immune response is taking place a few days after infection. Molecules that trigger the action of our lymphocytes during the adaptive response are called antigens. Originally, the work antigen is the contraction of two words ANTIbody-GENerator indicating that antigen are recognized by antibodies. Nowadays, this notion also applies to molecules recognized by T cell through their T cell Receptor. How the antigens are presented to T cells is a bit complicated: it follows an antigen presentation process performed by dendritic cells originating from the site of infection. During this antigen presentation, molecules critically involved are the famous molecules of the major histocompatibility complex (MHC), also called HLA molecules in humans and, by clicking on their name, you will discover the beautiful structure of the two main types of these molecules: HLA class I and HLA class II.
Structre of the MHC class I and MHC class II molecules

--> Two other films show you how antigen presentation by HLA class I and antigen presentation by HLA class II to T cells operates. Finally you can have fun identifying antigens yourself in proteins of your choice using the dedicated freeware SYFPEITH.

--> How can our T and B lymphocytes recognize all the microbes potentially present in our environment? Unlike cells of the innate immunity that have some PRR, each capable of recognizing many different microbes, T and B cells each have a receptor very specific for a molecular fragment of a given microbe. For B cells, this receptor is called BCR (B Cell Receptor), which is nothing else than an immunoglobulin anchored in the membrane of the B lymphocyte. For the T cell, the receptor is the TCR (T cell Receptor) which recognizes the antigen presented by MHC molecules on presenting cells. There are a large number of different T and B cells each carrying a different BCR or TCR that determines our ability to react against virtually any aggressor! One speaks of a "repertoire" of  B and T lymphocytes circulating in our blood to describe the extent of the diversity of T lymphocytes and B having different antigenic specificity.

Estimated size of the total repertoire of immunoglobulins and TCR
--> Generate such a directory is not obvious and involves an uncommon genetic coding system combining a relatively large number of gene fragments and a process of random recombination events between these gene  fragments yielding a huge diversity of possible combinations corresponding to highly diverse antigenic recognition capacities (see animation). As this process is random, this also means that within this repertoire, there are lymphocytes capable of recognizing our own molecules! These cells are very dangerous for us and there is a need to get rid of these dangerous cells, which is achieved by process called SELF tolerance. 
In fact the final reperoire of T and B cells is teh result of an equation:
Total repertoire - SELF-specific repertoire = NON SELF-specific repertoire!

Here we are! Many different pathogens can now be recognized very specifically and in a safe way!

--> For thos of you who can understand french, you will find a very interesting lecture by  Jean-Claude Weill on Canal-U explaining these notions in deeper details.

T lymphocytes and B work different than cells of the innate immunity to eliminate microbes. B lymphocytes produce immunoblog-on-line, uh .... should rather write immunoglobulins ::) (also called antibodies) which will bind to antigens either soluble or present at the surface of pathogens leading to their elimination.This involves cellular activities previously studied in the context of innate immunity such as phagocytosis. In this case, pathogens opsonized by antibodies are recognized very effciiently by phagocytes through receptors for the Fc portion of antibodies called FcR. In another context, eosinophils can destroy pathogens opsonized by IgE antibodies. There is also activation of the classical complement pathway leading to the destruction of bacteria. Antibodies can also simply neutralize the action of microbes or their toxins: it is this property which is often implemented by antibodies generated after vaccination! For example, antibodies can block bacterial antigens that target our cell receptors and thereby prevent the infection of our cells. They can also bind to bacterial toxins and prevent these toxins from reaching their targets on our nerve cells or muscle.

Functions of classes and sub-classes of antibodies
--> Finally, antibodies including IgE, also act by binding to the FcR present on mast cells and basophils, resulting in their activation: although it does not lead to an elimination of pathogens, it strengthens the immune response since, for instance, the activation of mast cell maintains inflammation. Many types of antibodies are produced during the immune response: there are IgM, IgG, IgA, and IgE (there is also IgD but these ones are not very much secreted): IgM, A, G, E, D are various classes or isotypes of antibodies (not to be confused with allotypes and idiotypes). These classes have different functions, especially as they bind to different FcR worn by different cells. In addition, they have different locations and abundances. To illustrate this point, IgG is the only class of antibody able to cross the placental barrier: they play a role in protecting the fetus during pregnancy as the immune system of the fetus is immature! IgA are found in breast milk: they protect the newborn as his/her immune system is not strong enough to protect it. Fetuses and newborns are protected against pathogens without having been vaccinated: it is termed passive immunization or passive vaccination because they receive antibodies produced by others!

Mechanism of CD8 T cell cytotoxicity
--> CD8 T cells have cytotoxic functions after being activated by dendritic cells. They can then exert those lytic functions against target cells onto which they recognize the antigen presented by MHC class I, thanks to their TCR, (which indicates that the cell was infected). It then uses the same weapons (perforin + granzyme) as for the NK cell. CD4 T T cells themselves do not usually have direct effector functions to eliminate pathogens, but, after being activated, they produce cytokines that enable the involvement of B cells and CD8 + T cells: they act as orchestrators of the immune response, hence their name "helper T cells". The antigen-presenting cells (especially dendritic cells) play an important role in activating T lymphocytes (both CD4 and CD8).

--> A key feature of the adaptive response is that it differs qualitatively and quantitatively depending on the pathogen infects us for the first time or upon re-encounter of the same pathogen: in the first case, we speak about a "primary" response and in the second, about a "secondarye response. The secondary response is faster and more efficient. The greater efficiency of the secondary response is true for both the dynamics of antibody response and the T lymphocyte response. Why? Let's look at this question in the case of B lymphocytes: when encountering a pathogen for the first time, our B lymphocytes do not know the pathogen: those B cells that will recognize the pathogen (those with the appropriate BCR, which are very rare ...) are called "naive." Because these cells are rare and unexperienced they will have to first divide to become numerous enough and equip themselves with weapons for pathogen destruction. The process whereby the B cells are chosen to proliferate is called clonal selection; the antigen "choses" thos clones of B cells pre-existing in the famous repertoire and able to recognize it through the BCR. These lucky cells can now proliferate! In the case of B cells, the anti-microbial weapons are mostly antibodies.

Dynamics of the antibody response

--> This process of clonal selection requires a certain time: this correspond to about one week during which these events are not easily discernible: one the one hand, they take place in the secondary lymphoid organ nearest from the infected tissue and not in the infected tissue itself; on the other hand, those cells are not yet capable of eliminating the pathogen and acquire this capacity with time. After this week, the B cells will produce antibodies, and even more of these antibodies:during the primary response IgM are predominantly produced. The antigen-antibody reaction can take place! These IgM poorly recognize and are relatively ineffective against the pathogen. By cons, little by little, some B cells replace their production of IgM by IgG (or IgA or IgE, it depends): this is a process termed class switching (or isotype switching). These new antibodies (mainly IgG) display a much higher affinity for the pathogen and are more effective. Part of B lymphocytes that are activated become memory cells: these cells live for a very long time (several decades): they usually have switched their antibody and remain in a state close to the activation. It is those cells that are responsible for the efficiency of the secondary response: indeed, if we re-infect with the same pathogen, these cells will come into action quickly since they are pre-activated (latency will be from shortened to 1-2 days only) and it will be more immediately effective as high affinity IgG are being produced. It happens almost the same for T cells except that their weapons are called perforin / granzyme or cytokines instead of antibodies. By the way, you have understood the immunological basis and significance of vaccination! The vaccine mimics the pathogen but without the toxic effects and when injected, the vaccine triggers a primary response of our immune system which leads to the generation of T and B cell memory: we speak of vaccination or active immunization. If you now encounter the pathogen mimicked by the vaccine, an intense and effective secondary reaction is triggered against the pathogen, which is eliminated faster! Clever, is not it? We find very old historical origins for vaccination, much before the way it works, as presented above, was understood!

Mycobacterium tuberculosis: the agent of tuberculosis
--> We talk a lot about immunity against infectious diseases and you understand that when things are going well, we can easily fight against most microbes. In some people, the immune system malfunctions: genes important for the immune response have mutations that prevent them from functioning normally. These are that patients with immunodeficiencies, who are very sensitive and may die of mild infections. This also happens when some microbes such as the virus causing AIDS (HIV: human immunodeficiency virus) attack our immune cells. If you want to know more about microbes, here are links to course hosted at the University of South Carolina, dealing with bacteria, viruses, parasites and fungi.

Plasmodium falciparum: the agent of malaria
HIV-1: the agent causing aids

--> Our immune system also allows us to fight against tumors. Many vaccines against cancers are being tested at the moment! In contrast to those positive role, our immune system also plays us tricks: if our immune system is poorly "educated", it can attack our own tissues: this can lead to auto-immune diseases. You can also have allergic reactions (or hypersensitivity) such as asthma, which is also due to the immune system.
Finally, we must take into account the immune system when you want to
transplant an organ to someone. The notion of NON-SELF does not only apply to microbes, but also to your brother or your sister or your parents, or whoever putative donor! That is why we are looking for a compatible donor for organ transplantation! The most compatible one is ...yourself (autograft) or your twin! In other cases, we must be careful and take drugs that weaken the immune system to prevent you from rejecting the transplant (Note: during this time you are very susceptible to germs!)

WANNA TEST IF YOU REALLY BECAME AN EXPERT IN IMMUNOLOGY? Test yourself with QUIZZ 1 and QUIZZ 2 proposed by the University of South Carolina, School of Medicine!

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