1. YOUR ACTIVE IMMUNE DEFENSES 
Innate Immunity
- invariant (generalized)
- early, limited specificity
- the first line of defense
Adaptive Immunity
- variable (custom)
- later, highly specific
- ‘‘remembers’’ infection
Use “enter” to advance each line in the animation.
1. Barriers - skin, tears
2. Phagocytes - neutrophils,
macrophages
3. NK cells and mast cells
4. Complement and other proteins

2. ADAPTIVE IMMUNE RESPONSE
a specific response
results in acquired immunity
long term immunity - “memory”
involves two types of lymphocytes:
T cells
B cells
T cells are formed in the bone marrow, develop in the thymus, provide help to and regulates B cells.
There are three main types. Helper T cells have receptors that identify foreign antigen, bound to the surface of specialized “presenting” cells, called antigen presenting cells or APCs. Binding antigen and another signal lead to their activation. When they are activated, the helper T cell secretes substances that stimulate B cell production, as well as the production of other T cells. Killer or cytotoxic T cells destroy specific cells invaded by pathogens by transferring specific proteins into membrane of pathogen and causing fluid loss. Suppressor T cells slow or stop production of B cells and other T cells when pathogens are destroyed.
B cells originate in the bone marrow. Each B cell will produce a different type of antibody and there are thousands of different B cells, so thousands of specific antibodies can be produced. B cells have the ability to reproduce themselves and form huge populations (clones), which circulate in the blood and lymph. When the B cells bind to their antigen, they proliferate and differentiate into plasma cells, which secrete antibody.
Some T and B cells activated by a specific antigen will remain specific and sensitized to that antigen, so that they can respond quickly when it might be next encountered. They live for a long time, even years, and will become quickly activated if their antigen is encountered again. This is called immunologic memory, and is the basis for the effectiveness of vaccines in protecting from certain infections.

3. ADAPTIVE IMMUNE RESPONSE
the specific response is customized for each pathogen
responsible for acquired immunity
involves antigen-presenting cells and two types of lymphocytes
turns on when needed - inducible
“remembers” the pathogens it has “seen” and goes into action faster the second time
may confer lifelong immunity
See teacher’s notes for the previous slide, number 2, entitled “Adaptive Immune Response”.

4. White Blood Cells (WBCs)
There are two main types of WBCs involved in the adaptive immune response:
antigen-presenting cells (APCs)
- not pathogen-specific
- ingest foreign substances and break them down
e.g., macrophage
lymphocytes
- pathogen-specific
- different types recognize different invaders and lead to their destruction

5. Human red and white blood cells
Human red blood cells (red), activated platelets (purple) and white blood cells - monocyte (green) and T lymphocyte (orange).
Colorized-SEM (scanning electron micrograph)
Magnification:-1200x--(Based on an image size of 1 inch in the narrow dimension)
Monocytes are WBCs that are typically round with a horseshoe-shaped nucleus. They are released into the blood and then migrate into tissues where they become macrophages. Functionally, they are phagocytic. Some macrophages simply ingest and destroy foreign material, others ingest, digest and then display fragments of the foreign material on MHC molecules (see notes for slide #8, “T cell training”. These macrophages are then called antigen presenting cells or APCs.
©Dennis Kunkel Microscopy, Inc.,

6. Types of lymphocytes
There are two types of lymphocytes. Both form from bone marrow stem cells:
T cells mature in the thymus
B cells mature in the bone marrow
Both cell types enter the lymph nodes and spleen after they are mature. From there they can look for foreign invaders in the bloodstream.

7. T cells
there are millions of different T cells – the difference is in their receptors (surface markers)
each T cell has a unique receptor that will recognize a different foreign substance
mature in the thymus, where they learn to tell the difference between self and “non-self”
- critical, because if they did attack “self”, autoimmune disease could result
The T cell receptor (TCR) is a glycoprotein with both constant and variable regions. The amino acid sequence of the variable regions is what allows the TCR to “recognize” specific antigen sequences. The gene rearrangements that allow for a large number of unique TCRs are very similar to those which allow for a large number of unique antibodies. In fact, it is thought that antibodies and TCRs may derive from a common ancestral gene.

8. T cell training
T cell precursors arrive in the thymus from the bone marrow
there, they express specific T cell receptors and meet cells that “wear” bits of self proteins, called MHC (major histocompatibility complex), that are markers for the body’s own cells
there are two steps
first, T cells must recognize self-MHC, or they are destroyed
in a second step, T cells that bind too tightly to self-MHC are also destroyed
remaining T cells go to the spleen and lymph nodes, and wait for antigens. If they recognize an antigen, some will “go into battle” and others become memory cells
MHC stands for “major histocompatibility complex,” which is a set of genetic loci that code for the MHC molecules that are an important set of cell surface markers found on immune cells.
MHC is also known as “HLA” in humans - “human leukocyte antigen”. Note that the term “antigen” in HLA is a little misleading - MHC (HLA) molecules are only antigenic when cells or tissues expressing them are transplanted into individual with another MHC type.
MHC molecules are the ones that phagocytic cells use to present bits of antigen to other immune cells. But MHC molecules also provoke the strongest rejection response in organ or tissue transplantation. Foreign MHC (Class I) can activate T cells, even in the absence of antigen presentation. As a result, tissue typing before a transplant is critical. This is done by serological testing, i.e., by mixing samples of tissue cells with preparations of antibodies against known MHCs. Getting a perfect match of all the myriad MHCs is virtually impossible, except in the case of identical twins. Not all MHCs are equally effective at provoking rejection, however, so testing for just one class (Class II) of MHC molecules is usually sufficient to get a good match. Recently, DNA testing has been used to tissue type.

9. Steps in T cell development
Step 1. Positive selection occurs in the thymic cortex
MHC self-recognition molecules
T cells only recognize antigen when it’s bound to MHC, so it is important for the immune system to select cells that recognize MHC.
Immune cells make a host of surface markers. Some are always expressed and identify the type of cell; some are only expressed during certain stages of development; and others are only expressed when the cell becomes activated. CD3 is a T cell marker that actually forms part of the TCR complex in a mature T cell. CD4 and CD8, also found on T cells, recognize MHC Class II and I molecules, respectively. T cells that have a marker are said to be “positive” for that marker, so T cells that express the CD4 marker are designated “CD4+”.
Immature T cells enter the thymus from the bone marrow. They begin their development in a section of the thymus called the thymic cortex, then move into the thymic medulla. When development is complete, they leave the thymus and enter the bloodstream or lymphatic system.
Immature T cells that have just arrived in the thymus do not express the usual T cell markers (CD3, CD4 or CD8). As they undergo development they first express CD3, which is expressed on all T cells regardless of their function, followed by both CD4, which is expressed in most cases on T helper cells, and CD8, which is expressed in most cases on T cells with cytotoxic or suppressor function. As the cells undergo selection, the levels of expression of these receptors changes so that the mature T cells that emerge all express CD3, but either CD4 or CD8, not both.
In the diagram, an immature thymocyte begins to express surface markers. These markers interact with thymic epithelial cells that express high levels of MHC. The epithelial cells present MHC to the immature T cells and those T cells that fail to interact undergo apoptosis. This is essential because T cells recognize antigen only when it’s presented to them on MHC, so failure to interact with MHC means that they will not be able to “see” antigen when it is presented. This process is called positive selection.
Note: The terms “CD4” may be familiar to students because of its relevance to HIV infection. The virus uses CD4 as a receptor to enter cells, and so “targets” CD4+ cells. The CD4+ cell count is used to monitor the progress of the infection. Immediately following infection, there is a sharp drop in the CD4+ count from around 1000/L to around 500/L, followed by gradual decrease over a period of years (about 8 on average) to levels approaching zero in the final stages. A diagnosis of AIDS is made when the count drops below 200/L. For additional information, please refer to Slide 31 in Lecture 3, entitled “Acquired immunodeficiency syndrome (AIDS)”.

10. Steps in T cell development (cont’d)
Step 2. Negative selection occurs in the thymic medulla.
Maturing T cells that survived positive selection in the cortex move into a region of the thymus called the medulla. At this point, some of these cells may have receptors that recognize “self” molecules other than MHC (recognizing self-MHC is good, but recognizing other self molecules isn’t). In the medulla, macrophages and interdigitating dendritic cells (specialized APCs in the thymic medulla) present bits of self-molecules or self-antigen to the T cells. T cells that recognize and interact with the self-antigen undergo apoptosis. Those that “ignore” the self-antigen survive and undergo final maturation. This process is called negative selection. After negative selection, T cells begin to express high levels of TCR and lose either CD4 or CD8, so that only one is expressed.
Fewer than 5% of thymocytes survive both positive and negative selection in the thymus. The “mess” created by the deaths of the apoptotic cells is “cleaned up” by macrophages.

11. Types of T cells
Based on function, there are different types including:
helper T cells – start the immune response
cytotoxic T cells – kill the body’s abnormal cells, like virus-infected cells and cancer cells
suppressor T cells – suppress the activities of other T cells, helping to end the immune response
Helper T cells (TH) are the first to interact with antigen-presenting cells or APCs. They have a variety of functions, including causing the activation of the antibody-producing B cells (which engage in “chemical warfare” by producing antibodies that react with antigen) and also activation of cytotoxic cells (which engage in “hand-to-hand combat” by producing chemicals which kill other cells). Usually these are CD4+ and recognize antigen presented on Class II MHC molecules.
Cytotoxic T cells (TC) are the body’s main defense against virus-infected cells and against cancer cells. When TC cells encounter a cell that presents foreign antigen, they may kill by literally punching holes in the membrane of the APC using perforin, a protein that inserts itself into the membrane of the APC and forms an open pore. TC cells may also produce proteins that act at the surface of the APC, but trigger internal biochemical changes that result in apoptosis of the APC. TC are CD8+ and recognize antigen presented on Class I MHC molecules.
Suppressor T cells (TS) are antigen-specific and suppress the functions of other types of T cells. The mechanisms by which TS cells downregulate the other cells are not well understood, but they include secretion of chemical signals (cytokines) that cause the other T cells to produce fewer of the surface markers that are involved in their activation. Suppression function is important when an infection has been contained and an immune response is no longer appropriate.

12. A Cytotoxic T Cell Attacking and Killing a Virus-Infected Target Cell
CELLS alive!
Here, the smaller cytotoxic T cell or Tc (arrow) is attacking and killing a much larger virus-infected cell. The T cell will survive while the infected cell is destroyed.

13. B cells produced and mature in bone marrow
each B cell produces and wears a unique antibody on its surface
clonal selection - when a B cell encounters a matching antigen, it begins to divide rapidly. Some then become plasma cells that all produce the same antibody, and then die. Others become memory cells.
the specific antibody produced by a plasma cell is also secreted in soluble form and circulates in the blood

14.   Selection of B cells by antigen (clonal selection)
Different types of B cells have different receptor molecules. 
When a pathogen (germ) “locks on” to a receptor, that type of B cell is selected.
Antibodies are produced by B cells. They are either displayed on the cell surface as markers or released into circulation as circulating markers. In order to have specific recognition of antigens by B cell antibodies, there must be different antibodies for different antigens.
In this slide, we simply assume that the diversity is there and that the specificity of the B cells is due to the unique antibodies that they display on their surfaces (each B cell “specializes” in making one particular antibody). Each antibody is capable of recognizing one unique antigen.
When a B cell’s antibody binds to its matching antigen, the B cell becomes “selected”. It expresses new receptors that allow it to receive chemical communications from other cells. It begins to secrete chemical signals itself and undergoes a number of division cycles. At some point, most of the dividing B cells undergo morphological as well as chemical changes and become plasma cells, with enormous amounts of endoplasmic reticulum allowing it to make large amounts of the specific antibody. The remaining B cells function as “memory cells” which are capable of responding quickly to subsequent infections; in fact, their response is much quicker than that of the original resting B cell. Plasma cells have a lifespan of only a few days, whereas memory cells may persist for many years.
The principle of vaccination is based on immunological memory. The initial vaccination introduces an antigen and provokes a primary response, which consists of the initial B and T cell selection step and activation. Subsequent exposures to the antigen activates memory T and B cells, and their response and production of antibody is quick enough to eliminate the antigen before disease symptoms occur. 
The selected B cell divides rapidly to make lots of copies of itself. The copies make lots of antibodies against the pathogen.

15. Antibodies specific – react with only one antigen
Are Y-shaped proteins called immunoglobulins (Ig)
each is made of two heavy and two light chains of amino acids, held together by disulfide bonds
Antibodies are made of proteins that bind to a specific part or determinant of an antigen called an epitope. Each epitope occurs many times, or is studded on the surface of a pathogen. The antibodies that are produced act as the functional units of the immune response. Shaped like a “Y”, the antibody has two sites at the tip of the Y which are the binding sites of the antibody for the antigen.

16. Antibody structure
Each is made of two identical heavy and two identical light amino acid chains, held together by disulfide bonds
parts of the antibody (Ab) are constant, i.e., the same for every antibody
- parts are variable - the arms of the “Y” have different amino acid sequences that cause specific binding to antigen
the fact that there are many different variable regions results in antibodies that react with almost any antigen you could possibly encounter!

17. Antibodies (Immunoglobulins)
Humoral response
Antibodies (Immunoglobulins)
Antibody molecules bind with great specificity and affinity to the antigen that originally activated the B lymphocyte
Each antibody molecule has two or more sites for binding antigen, so antigen molecules can be cross-linked, as in precipitation or aggluttination reactions
Antibodies play a number of essential roles in an effective immune response

18. Physical & Chemical Barriers
Antibodies (Immunoglobulins)
Roles of Antibodies
Precipitation: Clumping and precipitation of soluble antigens
Agglutination: Clumping together of cellular antigens
Virus neutralization
Toxin neutralization
Complement fixation: Antibody molecules can trigger a complement pathway leading to the lysis of a cellular antigen
Opsonization: Antibody molecules can coat a cellular antigen, making it much easier for a phagocyte to recognize and engulf

19. Antibody – another view
- variable regions of the light chain (grey) and the heavy chain (yellow) form the antigen binding site
- light chain constant region is blue while heavy chain constant region is red. The two chains are joined by carbohydrate (purple).
This is a space-filling model of immunoglobulin. Yellow regions in the heavy chain constant region represent amino acids that may be variable –they are not part of the antigen binding site.
©Mike Clark,

20. Four classes of secreted antibodies
IgM – a pentamer – five Y-shaped immunoglobulins joined together – the “early” Ab, it is produced before any of the other types – it activates complement
IgG – the most common form, and the major one for secondary responses
IgA – mostly a dimer – two Y-shaped immunoglobulins secreted in saliva, colostrum, milk, semen, mucus
IgE – binds to receptors found on mast cells – involved in allergy and parasitic infections
Antibodies function to form antibody-antigen complexes, which: 1) direct the enhancement of phagocytosis (opsonization or recognition); 2) direct the neutralization of antigens (such as toxins, viruses); 3) cause activation of complement system.
The classes of immunoglobulin differ in both structure and function. Differences in class are related to differences in the constant portions of the heavy chains. Production of unusually large quantities of one antibody class may be of value in diagnosing illness. For example, high titers of IgE may indicate a parasitic infection. IgG is the class of antibody shown in the preceding slides.
All B cells begin by making IgM, the “early” or first type of antibody, which they display on their surfaces,and also secrete. But they may later switch to form one or more of the other classes. This will involve a rearrangement of DNA in the constant region of the heavy chain DNA. (Switching classes results from signals from CD4+ TH cells.) B cells will also display another type of Ig on their surfaces, IgD. IgD does not exist in a secreted form.
After stimulation by an antigen, B cells continue to make the same variable portion –which contains the antigen-binding site – but may make a slightly different constant chain. They switch from making the “early” IgM to making IgG, which may be secreted and is the main immunoglobulin in the secondary response. The new antibody is still directed against the same antigen, but it is now soluble rather than membrane-bound, and also may have different functions.
A couple of vocabulary terms:
Colostrum is the clear “almost milk” fluid secreted by lactating females prior to actual milk production. The IgA present in the colostrum is coupled to a secretory component (actually part of the receptor which transported it across the cells lining the ducts in the mammary glands). This secretory component helps protect the IgA from digestion by proteolytic enzymes.
Mast cells are a type of auxiliary immune cell. They are recognized microscopically by the presence of large granules in their cytoplasm. When activated they release the granules (a process known as degranulation) and the chemical constituents of the granules cause inflammation and attract other WBCs to the site. Mast cells are located in close proximity to blood vessels. In the case of allergy, mast cells are activated when allergens bind to IgE molecules that are bound to the mast cell surface. One of the chemicals released during degranulation is histamine, which causes the symptoms of the allergy and is counteracted by pharmaceutical anti-histamines. In a normal response to a foreign substance, histamine produces a hostile environment for the foreign substance and assists in its elimination. (So, allergy is a case of an appropriate response to an inappropriate stimulus.)

21. Antibody response

22. Antigens Antigen (Ag) – the molecule an antibody (Ab) binds to
usually a foreign substance
each antigen has different sites that antibodies can bind to, so that one antigen can be bound by several different antibodies
examples in the case of allergy could be pollen, cat dander, or a chemical in soap
This figure is a molecular model of a pollen grain; different colors represent different antigenic determinants. Antigens are the foreign triggers for a very specific defense action of the human immune system. Your body reacts to these triggers in a primary response from your first exposure to a pathogen and/or a secondary response at a subsequent (later) exposure to the same pathogen by producing antibodies as a defensive action.

23. How Antibody Binds to Antigen
On this slide, the top panel of images is a schematic representation of the binding of antigen to antibody. The blue portion corresponds to the antibody molecule, with VH representing the variable portion of the heavy chain and VL representing the variable portion of the light chain, that come together to form the antigen binding site. The red dot (orange cylinder, yellow wedge) symbolizes a single determinant of an antigen. On the previous slide of the pollen grain, entitled “Antigens”, each dot on the pollen grain represents a potential antibody binding site. Complex antigens like pollen grains have many binding sites and can bind to more than one antibody. In fact, most antigens stimulate more than one type of B cell. But the antibody that each B cell produces is specific for only for its antigen.
The different orientations in the top panel of images simply represent some of the various spatial interactions that may occur between antigen and antibody. The lower panel of images represents the same interaction, but as a molecular model of the antigen/antibody interaction as might be seen if one were looking downward, directly into the antigen binding site.
The top part of this figure shows how different shaped antigens can fit into the binding site of antibodies: left, pocket; center, groove; right, extended surface. The panels below show space-filling or computer-generated models indicating where contact between the peptide antigen and antibody occurs.

24. How an Antibody Works
When an Ab finds its Ag on an invader, it will bind there and act as a “trash tag”, marking it for destruction by “killer” cells, macrophages or complement
Receptor for constant region of antibody on NK cell recognizes a bound antibody
After binding, the NK cell is signaled to kill the target cell
The target cell dies by apoptosis and/or membrane damage
Antibody binds to target antigen
When the variable (Fab) portion of an IgG molecule is bound to an antigen on the surface of a cell, its constant (Fc) region interacts with specific receptors on neutrophils and macrophages. In other words, the antibody forms a link between the antigen and the cell that will phagocytize it. The antigen is ingested and destroyed.
In the slide, antibody is bound to the target antigen. Binding of a single antibody is not enough to target a cell for destruction by an NK (natural killer) cell. In order for the NK cell to be activated, several Fc regions must crosslink with the IgG receptors on the NK cell. Requiring several binding sites for activation provides a check on the system and reduces the chance of errors.

25. The Number Dilemma
You have about a trillion different antibodies able to react with millions of different types of Ag
but you only have about 30,000-60,000 genes which code for all the proteins you need in your entire body, most of which are not Ab
so there cannot be one gene for one antibody to code for these – we wouldn’t have enough antibodies!
So how can your body produce Ab to so many antigens, even those it’s never seen?
This is a nice opportunity to review the connection between antibodies (proteins) and genes. The genetic mechanisms for achieving diversity are the subject of the next several slides.
Students have probably not thought about how large the number of potential antigens is, or about the need for so many different antibodies. The ability to generate a large number of antibodies is present in the immune system, waiting to be selected as needed. Since the number of antibodies an individual might need doesn’t correlate with the number of different genes devoted to antibody production, there is another mechanism, gene rearrangement, that generates the required diversity. This is definitely not “one gene – one polypeptide”.

26. Antibody Variability
There are several reasons why there are an enormous number of different antibodies:
different combinations of heavy and light chains which are encoded by different genes
recombination
others
There is more detail about this in the next few slides.

27. Antibody Genes
Genes for antibodies aren’t like most other genes - they come in pieces (“gene-lets”):
variable segments (V) – many different versions
diversity segments (D) – several different versions
joining segments (J) – a few different versions
constant segments (C) – a few different versions that are nearly identical
Note that the term “gene-let” isn’t accepted terminology, but it does convey the fact that we are talking about segments of genes, not whole genes.
Further information is on the following slide, entitled “A Unique Recombination Occurs in Each B Cell”.

28. A unique recombination occurs in each B cell
each B cell combines these gene segments to make an Ab chain like shuffling a deck of cards
- V, D, and J for the heavy chain, V and J for the light chain
since there are multiple types of each gene segment, there are many thousands of possible V-D-J combinations so that each B cell gets a unique combination of segments!
Unique combination of segments becomes joined by somatic gene rearrangement
Our understanding of antibody diversity has largely depended on DNA technology. The numbers of genes cited below apply to mice and are only approximate, and are likely to increase as the technology improves. No one really knows the numbers for sure at this point.
Each heavy chain is made from three smaller variable segments – V, D and J, and one constant segment – C. Each light chain is composed of two variable segments – V and J, and one constant segment – C. The V, J and C segments for heavy chains come from a different “pool” than the V, J and C segments for light chains. The two pools are on two different chromosomes.
In mouse “germ line” DNA, before there is any actual expression of antibody, the segments for each pool are arranged in clusters that are fairly far apart. During B cell development, a rearrangement occurs in each cell that is unique to that B cell. For light chain formation, one of the V segments, a J segment and a C segment are all brought close together. (Which V or J segment is chosen is a random event.) They are then transcribed as one chain, and translated resulting in the formation of a unique light chain. The same thing happens in heavy chain formation, except that a D segment is also involved in formation of the variable region.
In mice, there are approximately 300 V segments and 4 J segments for light chains. This means that 1200 combinations are possible for light chains alone. In the case of heavy chains, there are 500 V, 12 D and 4 J segments. This allows for at least 24,000 combinations for heavy chains alone. Since each antibody molecule consists of both heavy and light chains, there would be 1,200 x 24,000 combinations, even if no other processes to generate diversity were at work.

29. A unique recombination occurs in each B cell
each B cell combines these gene segments to make an Ab chain like shuffling a deck of cards
V, D, and J are joined to C for the heavy chain, V and J are joined to C for the light chain
Further information is on the preceding slide, entitled “A unique recombination occurs in each B cell”.

30. Since there are multiple types of each gene segment, there are many thousands of possible V-D-J combinations so that each B cell gets a unique combination of segments! Additional diversity occurs because there are two types of light chains.
This slide gives current best numbers for human antibody segments. You could do some simple calculations like those in the notes of slide 25, “A unique recombination occurs in each B cell” to determine how many combinations are possible based only on the number of different segments.
K and λ refer to two distinct forms of light chains that exist in most vertebrates. An IgG molecule may have two K chains or two λ chains, but not both.

31. Other sources of variability
when V, D, and J pieces are joined, they may not always be joined perfectly – if some base-pairs are lost or added, the Ab will end up with a different amino acid sequence
variable region genes mutate at a higher rate than other genes in your body
The mechanism by which segments that may have been widely separated on a chromosome are brought together during B cell development, called rearrangement, is not well understood. What we do know is that the segments that must be joined have flanking sequences that are not part of the code for antibody, but rather ensure that V, D and J are linked in the correct order and in the correct locations. Sometimes this system fails and one or more nucleotides are lost in the joining process. This may be minor and actually lead to increased diversity of functional antibodies, or it may be more serious and cause a frame-shift that results in a non-functional antibody.
Some of the adverse mutations that occur during B cell development are caught by a B cell selection process that looks at binding affinity. A few days after antigen stimulation, B cells pass through germinal centers in the spleen or lymph nodes. Rapid division of the B cells begins there, during which hypermutation of the immunoglobulin genes occurs. APCs in the germinal centers present antigen to the B cells. Those cells that bind tightly to the antigen survive and those that bind loosely or not at all undergo apoptosis. B cells that survive the selection process produce antibodies with high affinity for the antigen.

32. B cell memory

33. YOUR ACTIVE IMMUNE DEFENSES 
Innate Immunity
- invariant (generalized)
- early, limited specificity
- the first line of defense
Adaptive Immunity
- variable (custom)
- later, highly specific
- ‘‘remembers’’ infection
Use “enter” to advance each line in the animation.
1. Barriers - skin, tears
APCs present Ag to T cells
2. Phagocytes - neutrophils,
macrophages
2. Activated T cells provide help to B cells and kill abnormal and infected cells
3. NK cells and mast cells
3. B cells - produce antibody specific for antigen
4. Complement and other proteins