The Immune System

INTRODUCTION

The immune system is a diffuse collection of cells and organs that function to protect the body against antigen, which are foreign molecules that are potentially harmful. The two ‘arms’ of the immune system are the innate and the adaptive immune systems. These two ‘arms’ have different properties and functions, and they also have a lot of overlap, which will be covered in more detail later.

As previously stated, the immune system is not contained in one organ, but is distributed in various organs and tissues throughout the body. Primary immune organs are defined as places where B and T lymphocytes develop. This group includes the bone marrow, where hematopoesis occurs (and thus B and T lymphocytes are born) and B cells mature, and the thymus, where T cells mature (immature cells are located in the cortex, mature cells in the medulla). Secondary immune organs are sites where mature, but naive, B and T cells encounter antigen and develop into effector cells. In the spleen, which filters the blood, T cells are located in the peri-arteriolar sheathes of the white pulp, while B cells are in the follicles, germinal centers, and the marginal zones. In the lymph nodes, which filter the lymph, B cells are located in the follicles, and T cells are located in the parafollicular areas.

The immune system operates by some basic principles. One is that there is an element of specificity, as in each B and T cell is only activated by binding specific antigen that is not ‘self’. The immune system is capable of discerning what is ‘self’ and what is ‘non-self’, and thus may be a threat and should be attacked. Another basic feature is the ‘memory’ of the immune system. When antigen is encountered for a second time, the immune response is heightened and quickened compared to the first encounter.

The clonal selection theory can explain some basic properties of the immune system. Many, many B and T cells, each with a unique and randomly generated antigen receptor, are generated even before the body encounters antigen. This occurs by a process known as somatic recombination which will be covered later. When the body does encounter antigen, is has a randomly generated arsenal of B and T cells with many various receptors, just waiting. If antigen does happen to match one of the B and T cell receptors, the activated lymphocyte will expand (a process known as clonal expansion), and thus an army of cells will exist in the body specific for that certain antigen the next time the body encounters it. This explains how the immune system has its memory. The concept of MHC restriction ensures that T cells recognize both non self antigen only when bound to a self MHC molecule

THE INNATE ARM OF THE IMMUNE SYSTEM

The innate immune system can be thought of as the body’s first line of more non-specific defense, and the adaptive immune system is the more specific defense system involving T and B lymphocytes. However, even though we are dividing the immune system into two ‘arms’, they work together in many ways, and there are certain cells (such as gamma-delta T cells and CD5 B cells) that sort of have innate and adaptive characteristics. There is not really a clear, harsh

division; however, the innate and adaptive do exhibit some different characteristics. The innate immune system is more nonspecific and more quickly acting than the adaptive and has no immunological memory. The most basic component of the innate immune system is physical barriers to pathogens, such as skin and mucous membranes. It also includes chemical barriers such as stomach acid, lysosyme, mucous secretions, and antimicrobial peptides. If a pathogen manages to slip by the barriers of the body, the innate immune system orchestrates an inflammatory response. Phagocytic cells, inflammatory cytokines, and the complement cascade, are also included in the inflammatory response. Interferons, which are compounds produced by virally infected cells, and natural killer cells, which kill virally infected and tumor cells, are also considered part of the innate immune system.

Let’s look at the inflammatory response orchestrated by the innate immune system when a pathogen successfully gets past our barriers. We can learn about the type of pathogen causing the infection and how long it has been going on by the certain changes in the white blood cell profile of a patient

Neutrophils are the first on the scene, especially in a bacterial infection. There are large reserves of neutrophils in the bone marrow and they can be released as needed. When needed, neutrophils, and also other WBCs (dependent on the type and time course of infection) are activated to leave the bone marrow by inflammatory cytokines produced by activated macrophages, injured tissue, and mast cells. IL-8 specifically recruits neutrophils to tissues. IL-1, IL-6, and TNF-a are classic inflammatory cytokines released by macrophages, and they induce fever and promote WBC adhesion in the blood vessels, along with eliciting various inflammatory effects in a wide range of organs and tissues. When the neutrophils and other WBCs leave the bloodstream and enter the tissues they do so by a process known as diapedesis, which is rolling and squeezing through the endothelial cells. Diapedesis is made possible when cytokines induce expression of adhesion molecules on both the WBC and the vessel cell.

Once the neutrophils are out of the blood vessel and in the tissue, they engulf bacteria and die, and then they themselves are then cleared by macrophages. If there are a lot of immature neutrophils, called bands, this indicates that infection has been going on for a long time and the older neutrophils have already been exhausted. Other changes in the WBC profile can indicate infection. Monocyte percentage can increase in a more prolonged infection, and eosinophil percentage increases are typically seen with parasitic infections. If an infection continues, fever (due in part to bacterial pyrogens, IL-6, IL-1, and TNF-a), anemia (because of increased WBC production), and other organ changes can be observed.

Neutrophils and macrophages are just two examples of phagocytic cells, an important component of innate immunity. These phagocytic cells have evolved to recognize specific patterns that are indicative of pathogens, called PAMPs (pathogen-associated-molecular-patterns) through Toll-Like-Receptors (TLRs). Some examples of these PAMPs are lipopolysaccharide (LPS) in bacterial cell walls, mannose, double stranded RNA, single stranded DNA, and flagellin. Recognition of these PAMPs promotes engulfing and killing of the pathogens through the use of a phagolysosome. However, this does not always work, as in the case of mycobacterium tuberculosis, which can actually live inside of the phagolysosome. Engulfing of pathogens can promote phagocytic cells to make inflammatory cytokines, such as

IL-1, IL-6 and TNF-a. These cytokines promote inflammation in general, and will cause an accumulation of fluid, plasma proteins, and WBCs in the tissues.

The complement system is a series of serine proteases that can enhance the inflammatory response and phagocytosis of pathogens by cells in the innate immune system. It occurs in a cascade pattern, and has regulatory ‘brakes’ to inactivate the cascade when needed. There are two main phases of the complement cascade: activation and amplification. There are three complement pathways, and they are distinguished by their ‘activation’ phase: the classical pathway, the alternative pathway, and the mannose-lectin pathway. Although they all have different ways of being activated, they all converge and use the same set of molecules eventually. Specifically, they all form what is called a C3 convertase. We’ll talk about the alternative pathway here, and the classical pathway in the section on humoral immunity, because it needs antibodies to start it off.

The alternative pathway can be activated without any specific antibody. The main effects of this pathway are opsonization of pathogens by a molecule called C3b, lysis of pathogens by the membrane attack complex, and the release small proteins that are of mediators of inflammation.

Let’s first talk about how the molecule C3b ends up opsonizing pathogens. The alternative pathway it begins when a protein called C3 is spontaneously hydrolyzed at a very low rate in the blood, forming the fragments C3a and C3b. C3b will bind to a protein called factor B. When B binds C3b, a protease called factor D is now able to cleave B into its fragments, Bb and Ba. Bb remains associated with C3b to form C3bBb, which is a C3-convertase, meaning it is an enzyme which will cleave C3 at a much faster rate than its spontaneous hydrolysis. Now, C3b is hydrophobic, so it is attracted to cell membranes. If it happens to land upon a ‘self’ (human) cell, there are regulatory proteins which will thwart the cascade and prevent our own cells from being marked for phagocytosis. CR1 and DAF are two molecules that will prevent the formation of a C3 convertase on our cell surfaces when C3b is bound. C3 convertase formation can also be inhibited by factor I, with help from cofactor H, by cleaving C3b into its inactive form, iC3b, which will not form the C3 convertase. If C3b binds to a pathogen cell membrane, however, there are none of these regulatory molecules and the C3 convertase will be allowed to form, stabilized by a molecule called Properdin. If C3bBb, the C3 convertase, is allowed to form, it will rapidly cleave more molecules of C3, causing more and more C3b to be deposited on the cell (opsinization) and mark it for phagocytosis. Macrophages have the CR1 (complement receptor 1) which will allow them to recognize C3b repeats and phagocytose opsonized pathogen. C3b can also combine with bound antibody promoting antigens to stick to vessel walls.

A second effect of complement cascade is the production of mediators of inflammation. The small fragments created by cleavage of complement proteins, namely C5a, C4a, and C3a, act as mediators of inflammation and as anaphylatoxins. C5a is also a potent chemoattractant for neutrophils.

A third effect of the complement cascade is the production of the membrane attack complex (MAC). So we continue on…after the formation of C3 convertases of C5 convertases can form as follows: The C5 convertase in the alternative pathway is formed by binding C3b to the C3

convertase C3bBb to make (C3b)2Bb. C5b, produced by the C5 convertase, initiates the assembly of the membrane attack complex (MAC). C5b binds C6, C7, C8, and multiple C9s. This complex forms a pore which inserts into the pathogen cell membrane and causes lysis. The molecules CD59, Protein S, and HRF all prevent the MAC from inserting into host cells. If a patient is deficient in any component of complement downstream to the C3 convertase, they usually just are more susceptible to infections by Neisseria species of bacteria. However, if they are deficient in C3 convertase activity, the complement pathway will be seriously thwarted and they will have major immunodeficiencies.

ADATPIVE IMMUNE SYSTEM

The adaptive immune response involves B and T lymphocytes. As we’ve said before, it is intricately entwined with the innate immune response. However, the nature of it is very different. While the innate immune response is quick acting, the adaptive takes longer. The innate immune system has no ‘memory’ while the adaptive does. The innate immune response is nonspecific, and the adaptive is very specific. As stated, the adaptive immune response refers to the actions of B and T lymphocytes. Let’s first look at how they develop in the body.

B AND T CELL DEVELOPMENT – The transition from progenitor cell to naïve B/T cell

B and T cells are remarkable cells in that each one carries a unique receptor for a specific antigen. The receptors on the B and T cells are very similar. They are both heterodimer molecules, with the B cell having a heavy and light chain, and the T cell having an alpha and beta chain. Both the B and T cell receptors are made up of a series of immunoglobulin protein domains. Both receptors have a constant region, and a variable region, which binds antigen. However, the B cell receptor (which is actually a membrane bound antibody molecule, antibodies are secreted B cell receptors) binds to conformational epitopes, that is surface patterns, of antigen, while the T cell receptor binds to a short linear peptide from antigen bound to an MHC molecule. (More on MHC later).

B cells are born and develop in the bone marrow, in a specialized environment with help from stromal cells. T cells are also born in the bone marrow, but they develop in the thymus. The stages of lymphocyte development are marked by what type of immunoglobulin receptors they are expressing.

You may be wondering how the body can possibly have a B/T cell that binds to the millions of different antigens out there with the limited size of our genome. The answer lies in the process of somatic recombination. With somatic recombination, a single progenitor cell is able to give rise to a large number of daughter cells with a unique antigen receptor. To have this happen, mini-genes (termed V D and J) are rearranged to form the mature antigen receptor gene. This process occurs before the cell has ever encountered antigen, and it only happens in B and T cells. The first step in somatic recombination (and B/T cell development) is the rearrangement of the D and J mini-genes on the heavy chain (B cells) or beta chain (T cells). Next, the V mini gene is rearranged on the heavy chain or beta chain locus, forming the mature heavy/beta chain for the B/T cell receptor, with intervening DNA spliced out. Proteins called RAG1 and RAG2 assist with recombination of the mini-genes, and a protein called Tdt fills in the gaps created by

recombination with new nucleotides. The area of the receptor with the most variability is called the CDR3 region, and is located at the junction between the V and D mini-genes on the heavy chain locus.

This whole time the heavy/beta chain has been rearranging, it has been paired with a ‘stand in’ light/alpha chain. Once a heavy/beta chain has been successfully rearranged it signals the cell to inhibit additional heavy chain locus rearrangement on the other chromosome. (If, after 2 tries of rearranging a heavy chain, the cell is still unsuccessful, it dies). This is allelic exclusion, and it ensures that a single B or T cell will only express one type of receptor. Once the heavy/beta chain has been made, we have ourselves a pre-B or pre-T cell and cell division is triggered. Now the light/alpha chain can rearrange. This only involves a V and J mini-gene rearrangement (no D). The cell now has it’s successfully rearranged heavy/beta chain with a rearranged light/alpha chain. If this binds very strongly to self-antigen, a process called receptor editing takes place. The cell has arrested development and the light chain locus on the other chromosome undergoes rearrangement to give it another try. If, after this second rearrangement, the receptor still binds very strongly to self antigen, the B/T cell dies by apoptosis. This is negative selection.

An additional note about T cell development. T cells can eventually become one of two types of effector cells: CD4 or CD8 cells. CD4 and CD8 are co-receptors, and are expressed on the mature cell in addition to a T cell receptor. When the T cell is before the Pre-T cell receptor stage (when it will have a successfully rearranged beta chain and a ‘fake’ stand in beta chain), it is termed ‘double negative’, because it expresses neither the CD4 nor the CD8 co-receptor on it’s surface. Once the beta chain has been successfully rearranged, Pre-T cell receptor signaling causes the cell to become ‘double positive’, that is it expresses both the CD4 and CD8 co-receptors. Once the alpha chain has been successfully made, the T cell can have 3 fates. It can die from neglect if it does not bind to any MHC molecules on the cells in the thymus, it can die from negative selection if it binds to MHC with self peptide with a very high affinity, or it can live because of positive selection if it binds to MHC with self peptide with low affinity. If it is positively selected for, whether it binds to MHC class I or class II will influence whether CD4 or CD8 sticks around as the co-receptor (the other one is down-regulated). If it binds MHC-I, it will become a CD8+ T cell, and if it binds to MHC-II, it will become a CD4+ T cell. Positive selection ensures that T cells are MHC restricted, that is they will only recognize antigen in the context of it being bound to an MHC molecule. The end product of all of this is a naïve CD4 or CD8 T cell that goes out to the periphery. The end product of B cell development is a naïve B cell that goes out to the periphery.

We should note here that all of these rearrangements are a prime set up for transforming a cell into a cancer cell. Somatic recombination may cause an oncogenic mutation, and many leukemias and lymphomas, such as Burkett’s lymphoma, are associated with it. There are other diseases that it is relevant to talk about here. Bare lymphocyte syndromes cause patients to have a complete lack of CD4 T cells if they do not express MHC-II and a complete lack of CD8 T cells if they do not express MHC-I. AIRE (autoimmune regulator) deficiency can occur when there is a lack of expression of a certain transcription factor in the thymus. This transcription factor promotes thymic expression of many peripheral proteins, so many self-reactive T cells that are normally weeded out via negative selection are allowed to mature. These patients have various autoimmune conditions.

There is a class of T cells, called gamma-delta T cells that arise in the fetal thymus that have a fixed antigen receptor. They are prevalent in mucosal areas and may play a role in recognizing common microbes that we encounter at epithelial barriers. In fact some can recognize antigen even when it is not bound to an MHC molecule. They can be thought of as kind of a bridge between adaptive and innate immunity.

MHC

We have been referring a lot to the MHC molecule. Here is where we can talk about its nature in a lot more detail. Basically, the MHC molecule is a protein made up of immunoglobin domains expressed by all nucleated cells in the body. All nucleated cells express MHC class I molecules on their surface, and antigen presenting cells express MHC class I along with MHC class II molecules on their surface. The MHC-I protein is made up of one alpha chain and one beta portion, which is encoded for on another chromosome that serves to stabilize the molecule. The MHC-II protein is made up of one alpha chain and one beta chain. Note the similarities between the MHC proteins and the T and B cell receptors. T cells can only respond to antigen when it is ‘presented’ in the context of an MHC molecule. The two MHC molecules bind to antigen in different ways. MHC-I binds a 8-10 amino acid peptide tightly. MHC-II binds a 13-18 amino acid in a more wobbly fashion, with the edges hanging off. Pathogens that are inside the cell, like viruses, bind to MHC-I and activate CD8+ T cells. Pathogens that are outside the cell, like bacteria, are engulfed by APCs, are presented in MHC-II, and can activate CD4+ T cells.

The MHC (aka HLA- human leukocyte antigen) is encoded for by multiple genes. MHC-I is coded for by genes called A, B, and C, and MHC-II is coded for by genes DP, DQ, and DR. Since MHC-I is made up of only 1 alpha chain (the beta stabilizing molecule is encoded for on another chromosome) there is only one A, B, and C allele on each chromosome, but since MHC-II is made up of two chains, there are alpha and beta loci for DP, DQ, and DR on each chromosome. MHC-I and MHC-II are codominantly expressed on cells in the body. That means that whatever alleles you get from your parents, you are going to express. Unlike B cell and T cell receptors, a given MHC molecule is not specific for just one antigen; it has the potential to bind many.

MHC molecules do not undergo somatic recombination like B cell and T cell receptors. Diversity is achieved by the face that the MHC gene loci are the most polymorphic in our species. That is, there are so many different possibilities for MHC molecules among the human species, so we are very unlikely to come across a pathogen that NO ONE can recognize and will wipe us all out. However, although there are many different alleles for MHC gene loci, there is a linkage disequilibrium, in that the frequency of some alleles is higher or lower than expected. MHCs, or HLAs are what is typed during ‘tissue typing’ for transplants. The highly polymorphic nature of the MHC genes is why matches are so rare. There are some MHC alleles that, if you express them, you seem to be more at risk for certain infections and autoimmune diseases. Certain viral or bacterial antigens, termed superantigens, can bind to the T-cell receptor and MHC outside of the peptide groove, nonspecifically, and can activate a large and uncontrollable number of T cells, causing a dangerously huge immune response.

Now we can talk about how MHC-I and MHC-II actually get their antigen in the groove and are expressed on the cell surface. MHC-I begins in the ER of the cell, partially folded and stabilized by the calnexin chaperone. It then binds its stabilizing beta chain. It is then released from calnexin, and binds to other chaperone proteins including calreticulin as it becomes associated with the TAP transporter. TAP serves to deliver a peptide to the MHC-I groove that has been degraded by the large protease called the proteosome. The loaded MHC-I is then released from TAP and exported to the cell surface. TAP deficiencies result in bare lymphocyte syndrome, which was discussed earlier. (A TAP deficiency would result in no MHC-I produced, and thus an absence of CD8+ T cells in the patient).

MHC-II also begins in the ER, where it is stabilized by an invariant chain molecule, which binds in its peptide groove, blocking peptide binding while the MHC-II is still in the ER. It is packaged into an endosome, where the invariant chain molecule is cleaved, leaving a short peptide called CLIP in the peptide groove. Endocytosed antigens are taken into the endosomes and degraded, but can only get into the MHC’s groove in the presence of HLA-DM, which causes CLIP to be released. The loaded MHC-II can now be exported to the cell surface.

T AND B CELLS ACTIVATION BY ANTIGEN - The transition from naïve to activated B/T cell

B and T cells become activated when antigen (or in the case of T cells, antigen in the context of MHC) binds to their receptors and a signal is transmitted as a result of that. This signal is initiated and propagated by tyrosine kinase activity. However, the T and B cell receptor themselves do not have any intrinsic kinase activity to them, so they must associate themselves with other molecules that do. The result of T and B cell receptors binding antigen is thus a signal is initiated, which begins with tyrosine phosphorylation of various proteins, and may involve second messengers such as calcium (important in B cell activation, resulting from the phosphorylation of phospholipase C and initiation of the IP3/DAG pathway) and G proteins, and eventually changes occur within the cell. The signal transduction pathway depends on protein-protein interactions that recognize certain domains (such as SH2 and SH3) within each other. Lipid rafts in the plasma membrane help to cluster signaling molecules together so they are more likely to associate with each other. When cells come together to initiate a signaling cascade it is termed, an ‘immunological synapse’.

Both the T and B cell are associated with invariant molecules which get their tyrosines phosphorylated when antigen binds the BCR or TCR. For the T cell, the invariant molecule is CD3, and for the B cell the invariant molecule is Ig-alpha/Ig-beta. The molecule that phosphorylates the invariant chain is a Src family kinase. The domain on the invariant chain that gets phosphorylated after ligand binding is called an ITAM. The Src family kinase that does the phosphorylating is associated with the co-receptor molecule in the B and T cell. In T cells, the co-receptor is either CD4 or CD8 and the Src family kinase associated is called Lck. After Lck phosphorylates the cytoplasmic tail of the CD3 invariant molecule, Zap70 is recruited to continue on with the signaling cascade. In the B cells, the co-receptor is a complex between CD19, CD21, and CD20, and the Src family kinase associated is called Lyn. After Lyn phosphorylates the cytoplasmic tail of the CD3 invariant molecule, Syk is recruited to continue on with the signaling cascade. Src family kinases are regulated by phosphorylation and dephosphorylation of tyrosines within certain regions. Csk is a tyrosine kinase that

phosphorylates an inhibitory tyrosine in Src family kinases, and CD45 is a tyrosine phosphatase that dephosphorylates an inhibitory tyrosine in Src family kinases.

Once the invariant chains in T and B cells are phosphorylated, the signal can be passed on through second messenger signaling. An example of this is the phospholipase C/IP3/DAG pathway, which creates an increase in intracellular calcium. This causes calcuneurin to localize the transcription factor NFAT to the nucleus to influence gene transcription. The drug Clyclosporin A blocks the calcium signaling pathway by inhibiting calcuneurin. Other second messenger systems that can be initiated by activation of protein kinase c, or through G protein signaling.

All of this has been about what B and T cells when they bind antigen. Naïve B and T cells move from the blood circulation (where they are unlikely to encounter their one specific antigen) to the lymph circulation, where antigen is concentrated in the lymph nodes and they have a better chance of being activated. Lymphocytes enter the lymph nodes from the blood through high endothelial venules (HEVs). Adhesion molecules, such as the ICAM/LFA-1 pair help to assist the diapedesis of the cell into the lymph node. If the cell does not encounter anything in the lymph node that bind to its receptor, it leaves the node through the efferent lymphatics, and will ultimately re-enter the bloodstream through the thoracic duct.

T cells are activated not by free antigen, as B cells are, but by antigen presenting cells that display a short peptide in their MHC molecule. For a T cell to be activated by an APC, it receives 2 signals. The first is the signal through the TCR binding the MHC-peptide on the APC. The second signal is the CD28 molecule on the T cell binding the B7 molecule on the APC. Binding of the co stimulatory molecule CD28 on T cells causes IL-2 production by the T cell and clonal expansion. Cytokines that are around at the time of naïve T cell activation can determine what kind of effector T cell it will develop into.

For a B cell to be activated, it also receives 2 signals, like the T cell. The first signal is the cross-linking of the B cell receptor by bound antigen. The second signal for the B cell is through the co-stimulatory molecule CD40, which binds CD40L on the T cell (specifically the Th2 cell, but we will get to this later). Other antigen presenting cells have the CD40 molecule, and can also be activated by T cells (such as macrophages). Thus there is reciprocal action between the B and T cells (and also between other cells in the immune system…this is one way that the adaptive and innate immune systems are related to each other!).

A B cell can also receive its second signal in a T-independent way, such as recognizing a PAMP through the Toll-like receptor. This will cause the B cell to differentiate into a plasma cell and produce a soluble form of its receptor called IgM. There are two types of T-independent antigens that are capable of activating B cells. The first, TI-1 antigens, are often microbial surface marker, like LPS, are also termed mitogens. These TI-1 antigens will cause many different B cells to become activated and begin production of IgM (polyclonal activation). However, B cells activated in this way will not undergo isotype switching (which will be covered later) and germinal center formation. TI-2 antigens usually have repeating domains, such as a bacterial polysaccharide capsule. However, the name TI-2 may be a misnomer, because some T cell cytokines may actually be involved for B cell activation by these antigens. IgG as well as

IgM can be produced by plasma cells that were activated in response to TI-2 antigen. IgG is very important for the opsonization of bacteria, which macrophages can bind with their Fc receptor and engulf. It is important to note that lack of any co-stimulatory signals in both B and T cells can result in tolerance.

CD5 B cells and Marginal Zone B cells are important subsets of B cells that often respond to antigen in a T independent manner. More about these in the section on B cell activation.

HOW T CELLS CARRY OUT THEIR FUNCTION ONCE ACTIVATED- The transition from activation to effector T cells

Once naïve T cells are activated through the TCR and CD28 in the lymphoid organs, they can have a few effector functions. CD8+ T cells have a cytotoxic effector function, basically killing cells infected with pathogen. CD4+ T cells can either act as Th1 or Th2 effector cells. There are mechanisms in the body that function to down-regulate T cell function to prevent immune over-reaction.

As a T cell (or a B cell for that matter) goes through development and activation the molecules expressed on the surface of a can tell a lot about what stage it is in. For example, a naïve T cell is likely to express L-selectin on its surface, which will cause it to be attracted to the HEV on the lymph node so that it can find antigen. On the other hand, an armed effector T cell is likely to express the adhesion molecule VLA-4 on its surface, which will cause it to be attracted to vascular endothelium so it can enter infected tissues and carry out its function. Once naïve T cells have a been activated they can carry out their effector function in a variety of ways, including cytokine production, the use of cytotoxins, and direct cell to cell contact. This transition from naïve to an effector T cell takes days, not minutes!

First, let’s talk about the possible effector functions of the CD4+ T cell. A naïve CD4 T cell can develop into a Th1 cell, which functions to activate macrophages, produce inflammatory cytokines, lyse cells, and help B cells produce opsonizing antibody. On the other hand, CD4+ T cells can also develop into a Th2 cell, which functions to help B cells produce antibody, and to help B cells through the production of cytokines. In a nutshell, Th1 cells will basically help macrophages with the destruction of engulfed pathogen, and Th2 cells will basically help B cells with the destruction of extracellular pathogen.

It makes sense then, that whatever pathogens are around will help influence whether a naïve CD4 T cell becomes a Th1 or a Th2 effector T cell. When there are engulfable pathogens, like a virus or bacteria, dendritic cells produce IL-12, which induces NK cells to produce IFN-gamma. IL-12 and IFN-gamma will influence naïve T cells to develop into Th1 cells. When there are pathogens like worms that are not usually engulfed, IL-4 production may be induced from some cells. This IL-4 will cause a naïve T cell that has just been activated to develop into a Th2 cell. These cytokines have cross regulatory activity. That is, IL-12 and IFN-gamma do not only promote Th1 development, but they inhibit Th2 development. IL-4 does not only promote Th2 development, but it inhibits Th1 development. As you would expect, Th1 and Th2 cells secrete a different profile of cytokines which are related to their effector function.

There is clinical relevance to Th1 and Th2 cells. Cytokines made by Th2 cells such as (IL-4 and IL-5) can induce many of the symptoms that mediate allergic reactions and asthma. Thl can sometimes become chronically activated when there is an engulfed pathogen that is hard to get rid of, such as mycobacterium tuberculosis, which lives inside macrophages once it has been engulfed. Basically, the Th1 cells continually, through production of inflammatory cytokines and through the CD40/CD40L interaction, activate more and more T cells and macrophages which can lead to chronic inflammation.

Another clinical example involving Th1 cells is the delayed type hypersensitivity reaction commonly seen with poison ivy. Lipid soluble poison ivy antigen can penetrate the skin and react with proteins, altering them. These altered proteins can be taken up by antigen presenting cells in the skin (macrophages and Langerhans cells) which present the antigen (derived from the altered proteins) to T cells in the lymph node. Upon the first exposure, a patient will not usually notice any symptoms. This stage is T cell priming. Upon subsequent exposure, Th1 cells that were primed will mediate an inflammatory response in the subcutaneous region. This response can take a few hours, because it is usually a small area and the Th1 cells that recognize this antigen are relatively rare. The inflammatory response is mostly caused by Th1 cells carrying out their effector functions, including inflammatory cytokine release, cytotoxicity via the FAS/FASL interaction (the expression of the FAS-ligand by T cells can bind with the FAS receptor on another cell, causing apoptosis of that cell), and production of degradative enzymes.

Now, let’s switch gears and talk about the effector functions of CD8+ T cells. Unlike CD4+ T cells which can have a ‘choice’ of whether to differentiate into Th1 or Th2 cells, naïve CD8+ T cells will develop into cytotoxic T cells when activated. Cytotoxic T cells basically carry out their effector function by killing their target cell, which makes sense when we remember that they are going to be activated by MHC-I molecules, which are expressed by all nucleated cells of the body. Since most cells in the body can’t carry out phagocytosis, if they have a pathogen inside them, it is something that infected them, such as a virus, and they should be killed. There are a couple of mechanisms of cytotoxicity that these effector cells use. The first is granule release. Perforin is released first from the cytotoxic T cell, which forms pores in the target cell membrane, allowing the granzyme granules to enter the target cell. Granzyme is a serine protease that activates the target cell’s apoptotic pathway. These granules are released at the site of cell contact. The second cytotoxic mechanism is the interaction of FAS-ligand on the T cell with FAS on the target cell, also serving to activate the target cell’s apoptotic pathway.

There is an additional subset of effector T cells that are neither Th1, Th2, nor cytotoxic, but they have a regulatory function. Mainly they serve to down-regulate effector T cell development and function. They can have direct contact with antigen presenting T cells and T cells to carry out their regulation, or they can produce cytokines that cause this down regulation. Often, these cells are CD4+ and CD25+.

Fortunately for us, T cell activation will not be allowed to go on forever in the body. When antigens are not present anymore, effector T cells that have been activated by that antigen will die by neglect. If there is antigen that is not being taken care of by the immune system and it is around for a prolonged period of time, the repeated stimulation of effector T cells can cause death via binding the T cell’s FAS receptor (by another cell’s FAS-L, or even the same cell’s

FAS-L, because there is so much activation, and thus so much FAS-L going on). If there is a deficiency in the FAS receptor, a disease called autoimmune lymphoproliferative disease (ALPS) results. These patients experience lymphadenopathy, lymphocytic infiltrates is organs, and autoantibody production.

Also, just as there are positive co-stimulatory molecules that provide a second signal for T cell activation (such as the B7 molecule, which binds CD28 on the T cell), there are negative co-stimulatory molecules. A B7 can also bind CTLA-4 on the T cell, causing down-regulation. PD-1, and some Fc-gamma receptors on the T cell are also molecules expressed on the T cell important in down-regulation of the immune response.

HUMORAL IMMUNITY –The transition from activation to effector B cells

After B cells have been activated by antigen and a second signal from T cells, they are stimulated to proliferate and differentiate into plasma cells. Proliferation occurs in germinal centers of the lymph nodes, sites of intense proliferation of B cells that are surrounded by the Th2 cells that activated them. In these germinal centers, B cells with better and better antigen affinity are produced via a process called somatic hypermutation. This process occurs only in B cells in germinal centers, and involves a high level of mutation in variable regions (V, D, and J mini genes) of the Ig gene. The B cells with increasingly good antigen binding capabilities are selected for, and higher and higher affinity B cells for the given antigen are ultimately produced (affinity maturation).

Ultimately, some B cells from the germinal centers will terminally differentiate into plasma cells. The Th2 cell induces plasma cell differentiation by CD40L binding and secretion of the cytokines IL4,5,and 6. Plasma cells are a terminally differentiated cell which no longer divides or responds to antigen. They no longer express membrane bound Ig (the B cell receptor) or MHC-II but they secrete Ig. They are larger than B cells, and have more ER, ribosomes, and golgi, representing their secretory function. Some B cells will terminally differentiate into memory B cells, which can be re-stimulated by antigen, providing immunological memory. Antibodies produced by plasma cells can bind Fc receptors on various other cells and cause a variety of things to happen, including but not limited to: promotion of phagocytosis of opsonized pathogen, neutralization of toxic pathogens, activation of cell killing through the ADCC (antibody dependent cellular cytotoxixity), and starting off the classical complement pathway.

We can talk here about the different terminology for antibodies, which are produced by plasma cells. Two antibodies that are isotypes have different Fc regions, which determines what the antibody will do after it binds antigen. Two antigens that are idiotypes have different FAb regions, which means that they bind different antigens. Two antigens that are allotypes are the same except for the small genetic difference between two people.

Plasma cells can produce antibodies of various isotypes. Different isotypes of antibodies have different functions. IgM is important in the primary immune response, are typically low affinity, is involved in complement fixation, and exist in serum as a pentamer. IgG is important in the secondary immune response, is an important opsonin, is involved in complement fixation, toxin and viral neutralization, and is able to cross the placenta. IgA is involved in preventing

microbial attachment to mucous membranes, is a dimmer in secretions, and is not involved in complement fixation. IgE mediates the immediate hypersensitivity reaction when it binds to the Fc receptors on mast cells and basophils, and it is a main defense against parasitic worms. IgD is found on naïve B cells and in low levels in the serum.

Once a B cell has differentiated into a plasma cell, it can not change the isotype of Ig it secretes. However, during proliferation of B cells in the lymph node germinal centers, isotype switching can occur. What isotype is switched to depends on what cytokines are in the microenvironment. Cytokines from Th2 cells induce what type of isotype switching occurs. Il-4 promotes switching from IgA to IgE. IL-5 stimulates switching to IgA. IFN-gamma promotes switching to IgG.

CD5+ B cells, or B1 cells, are a subset of B cells that have a high level of surface IgM and a low level of IgD. These, like gamma-delta T cells, can be kind of thought of as cells which exhibit characteristics of the innate and the adaptive immune systems. These develop in the fetus, and their receptor undergoes no somatic hypermutation, so they have a limited range of specificities. They seem to be able to recognize common bacterial antigens, and do not need T cell help to become activated. Abnormal proliferation of these cells is common in some types of leukemia.

Another notable type of B cells is the marginal zone B cells. These exist in the marginal zone of the spleen, and are responsible for most T-independent type 2 responses (TI-2). They can also be good APCs for T cells and function in T dependent reactions.

We talked about complement (the alternative pathway) in the section on innate immunity. Antibodies from plasma cells can activate another complement pathway, called the classical pathway. The classical pathway involves nine major protein components, activated in the following order: C1 (which has parts C1q, C1r, C1s), C4, C2, C3, C5, C6-8, C9. Both the classical pathway and the alternative pathway form a C3 convertase. The difference is how they get to the formation of that C3 convertase. (The mannose binding lectin pathway is another way to generate a C3 convertase.)

The pathway begins with the binding of the C1q protein to the Fc region of IgG or IgM, after it has bound antigen. It takes at least 2IgGs but only 1 IgM (because it is a pentamer), to start the pathway. (IgA, IgE, and IgD cannot bind C1q). C1q binding to antibody activates the protease function of C1r, which activates the protease function of C1s. C1s cleaves C2 and C4 into C2a (which can be converted to C2 kinin, a BV dilator) and C2b, and C4a and C4b. C4b covalently binds to a pathogen surface, where it binds C2b, forming C4b2b, which is the classical cascade C3 convertase. This C3 convertase can cleave C3 into C3a and C3b. C3b joins the C4b2b molecule to form C4b2b3b, the classical pathway C5 convertase. The rest of the pathway from this point on is the same as the alternative pathway. C3b and C4b can act as an opsonins (the CR1 on phagocytes will bind to them). Just like in the alternative cascade, the MAC can form. C3a, C5a (a potent chemoattractant for neutrophils), and C4a can all act as anaphylatoxins.

Many cells have receptors for components of the complement cascade. CR1 on phagocytes can bind C3b and C4b opsonins, causing phagocytosis of pathogens. There is also a receptor for C1q, which promotes phagocytosis of immune complex. CR2 (CD21) is a B cell co-receptor.

Binding of antigen-complement to CD21 increases sensitivity of the B cell to antigen by about 1000 times. Epstein Barr virus uses CD21 to enter B cells, and can occasionally cause a cancerous transformation. CR3 and CR4 allow monocytes, macrophages, neutrophils, and APCs to extravate into tissue. Mast cells and smooth muscle have receptors for C3a, C4a, and C5a.

Isohemagglutinins are naturally occurring IgM antibodies that exist prior to antigen exposure. An example of their relevance is blood type. All RBCs have an H substance, which is different in people of different genotypes . People with type A blood have antibodies against H substance type B, type O people have antibodies against H type A and H type B, etc. This is because we will view whatever H substance we do not have as a non-self antigen. So, it is very important to get the correct blood type in a transfusion, or a massive immune reaction to the ‘non-self’ antigen can occur.

Another clinical example of the importance of humoral immunity is the Rh antigen and pregnancy. Rh negative mothers can develop antibodies against Rh antigen if they are carrying an Rh positive baby. This is potentially dangerous to the baby if not assessed and treated, since the antibodies in this case are IgG antibodies which can cross the placenta. To prevent fetal harm, Rhogam is an Rh antibody that is given in very low does prophylactically to the mother. It is the same molecule that the mother would produce in her immune reaction, and it is given in low enough doses that it will not harm the baby. This low dose administration of Rhogam prevents the mother from making a large amount of her own Rh antibody. To test if a mother is Rh positive or negative, the Coombs test can be done. A sample of the mother’s serum is mixed with a control Rh positive blood sample. Coagulation indicates the presence of Rh antibodies, and indicates that the mother is Rh negative.