C2006/F2402 '11 -- Outline for Lecture 24

(c) 2011 Deborah Mowshowitz . Last updated 04/29/2011 11:00 AM.  

Handouts:    24A = Cells involved in immune system; clonal selection (Sadava fig. 42.7)
                    24B  = Top: Interactions of B & T cells; Bottom:  Antibody Structure (Sadava Table 42.3 & Fig. 42.9)
                    24C = T cell interactions and activations/killing of target cells (Sadava fig. 42.13)

Handouts are posted on Courseworks. Some problems to do have been added below. For a complete list of immunology problems, see the problems-to-do page.

I. Specific (= Adaptive) Immune Response -- Major cells and Features

We have already discussed antibodies as chemical reagents. How do antibodies, and the entire immune system, really work  physiologically? Sadava, Chap. 42 (18)

    A. Specific Immune system has 2 branches

1. In both branches:  

a. Cells make a specific protein that binds to a foreign substance. Foreign substance = antigen.

b. Protein and antigen match up like ligand and receptor (or enzyme and substrate).

c. Binding of specific protein to its target antigen is specific, and usually leads to destruction of target.

2. Humoral response

a. Specific cell protein is an antibody.

b. Antibody is released from cell that makes it.

c. Why 'humoral?' Binding and destruction of antigen done by proteins in "humors" = antibodies in blood and secretions (for ex. milk, tears).

d. Example:

(1). B cells → release antibody

(2).  Ab (antibody) binds Ag (antigen -- usually on surface of microbe)

(3). Binding triggers destruction of microbes (microbes are engulfed by phagocytes or lysed) often with the help of a set of proteins called complement.

e. Allergies are a side effect of this system. (See Sadava fig. 42.18 (18.19) if you are curious about the details)

3. Cellular or cell-mediated response

 a. Specific cell protein is on surface of T cells, not released.

b. Protein is called a TCR (T cell receptor).

c. Why 'cell-mediated?' Binding and destruction of antigen done by whole cells bearing a TCR.

d. Example:

(1). T cells → TCR on surface

(2). TCR's (of cytotoxic T cells) bind to Ag on surface of virus-infected eukaryotic cell

(3). Binding destroys target cell by triggering apoptosis (programmed cell death). 

e. Graft rejection is probably a side effect of this system. This is probably why grafts fail -- foreign cells of graft look like infected (defective?) cells and are destroyed. 

4. Big difference between the two branches -- Location of Target (as well as specific protein)

a. Humoral Response. Antibody (B cell protein) binds to antigens in solution or on surfaces of bacteria & viruses. Neither the protein mediating the immune response (antibody) nor its normal target (antigen) are on eukaryotic cell surfaces.

b. Cell-Mediated Response. TCR binds only to antigens on surfaces of other eukaryotic cells. Both the protein mediating the immune response (TCR) and its target must be on eukaryotic cell surfaces.

    B. What Cells are involved?  What are B cells and T cells? See handout 24A. White blood cells (leukocytes) -- contain no hemoglobin. WBC divided into two main types

1. Phagocytes -- macrophages, dendritic cells, etc. ( See Sadava  fig. 42.2 (18.2)). Involved in processing antigens so lymphocytes can respond to them, and/or engulfing (& destroying) antigens identified by the immune system.

2. Lymphocytes. Found in lymph nodes and elsewhere. Do actual production of antibodies and/or execution of cellular immune response.

a. Divided into B and T cells.

(1). Both B & T cells come from same line of stem cells in bone marrow.

(2). B cells mature in bone marrow; T cells in thymus

b. Role of B cells -- produce & secrete antibodies. Major players in humoral response.

c. Role of T cells -- Needed for cell-mediated responses. Two types

(1). Helper T's (T_H) -- Required for function of both T_C's and B's .

(2). Cytotoxic T's (CTL or T_C ) -- Kill target cells.

For an overview of both parts of the immune system, and the roles of B,  T_H & T_C  cells, see Sadava fig. 42.6, 9th ed. only.

    C. Table: Summary of Major features of 2 branches of specific immune system. Any features not covered yet will be covered later; this is here for reference. See notes after the table.

Notes:
*T cell receptor is NOT the receptor for T cells -- it is the protein on the T cells that is the receptor for an antigen. It is the receptor of T cells, not the receptor for T cells. 

** Complement = a series of proteins found in blood. Activation of complement involves a cascade of activations similar to that involved in blood clotting. Complement binds to antibody-antigen complexes attached to microbes and triggers phagocytosis or lysis of the microbe bearing the complex.

^# Antigen must be attached to a euk. cell surface protein called MHC. (Details later.)

^##Cytotoxic T cells use proteins called perforins to make holes in their targets. Then other proteins enter the holes and trigger apoptosis.  Note complement is similar to perforins but works on prokaryotic invaders; perforins work on rogue eukaryotic cells. Many texts say perforin lyses cells -- it makes holes in membrane, and then water enters, causing cells to swell and burst. (This is the way complement kills bacteria.) Newer data indicates perforin works to trigger apoptosis.

    D. What are the Important Features of the Adaptive Immune Response that need to be explained?

1. Specificity & Diversity -- each Ab or TCR is directed against one epitope or antigenic determinant [= piece of antigen], and there are many, many different antigens. How can you make so many different Ab's or TCR's, each specific for a particular antigen or piece of it?

2. Memory -- secondary response is faster, larger, better than primary response. In secondary response, make more Ab, Ab is more effective (binds better to Ag because of slight changes in amino acid sequence of Ab), and Ab response lasts longer. How is this done?

3. Tolerance -- can distinguish self/nonself or normal/abnormal -- make Ab only to foreign/abnormal stuff (except in disease states). TCR only directed against infected cells, not normal ones. How does this work?

4. Response is adaptable -- response depends on amount and type of antigen (& history of previous exposure). How do you "know" which antibody (or TCR) to make in response to a particular antigen?

5. How do helper T cells fit in? How do helper T's and cytotoxic T's distinguish their targets?

II. Clonal Selection -- How do you account for the "important features" listed above?

    A. B cells (See Handout 24A bottom = Sadava fig. 42.7 (18.6)

1. Each cell differentiates → produces a single type of Ab on surface (Cell called a "virgin" or "naive" B). Each cell makes a unique antibody -- that is, with a unique set of "grabbers" due to rearrangement of genes for antibodies.

2. Ab on surface of cell acts as a "trap". Surface antibody acts as trap/receptor for Ag. (Surface antibody also called BCR or B cell antigen receptor in parallel to terminology for TCR.)

3. Activation or destruction of B cell is triggered by binding of Ag to surface Ab (BCR)

a. Destruction. If Ag is perceived as "self" → cell destroyed or suppressed (→ tolerance).

b. Activation. If Ag is perceived as foreign → cell divides → clonal expansion, further differentiation into

(1). Effector cells (plasma cells) -- short lived but secrete lots of Ab → destroy or inactivate targets; class of Ab determines fine points. (In earlier lecture we explained how alternative splicing can allow cell to switch from making surface bound Ab to secreted Ab.)

(2). Memory cells -- long lived and more specialized to make Ab; wait for next time (responsible for memory). 

c. Whether antigen is perceived as "self" or "foreign" depends on time of exposure to the antigen (embryonic vs adult) and additional factors. (This turns out to be very complicated, so we will ignore the "additional factors.")

4. What's the point?

a. Clonal Selection: Each cell makes a little Ab before any Ag present. Each cell makes a different Ab. This antibody stays on the cell surface and acts as BCR = trap for antigen. Ag acts as a trigger -- binding of Ag to "trap" stimulates only those cells that happen to make Ab that binds to that particular trigger.

b. Clonal expansion: The cells triggered by binding of Ag grow and divide →  (more) effector cells  & memory cells . Both types of cells make only the antibody that binds to the trigger Ag.

c. Clonal suppression: The cells triggered by binding of self Ag are destroyed or suppressed (prevented from multiplying &/or making Ab.)

d. What does this explain?

1. Clonal selection is the part that accounts for specificity, diversity, and adaptability. How you make the 'right' antibody at the right time.

2. Clonal expansion and suppression are the parts that account for memory & tolerance -- memory when Ag triggers expansion (as in b) , and tolerance when Ag triggers destruction or suppression (as in c) .

5. Why do you need helper T cells? For most antigens, helper T must bind to B cell-Ag complex in order to activate B cell. (B cell must be activated to secrete Ab.) More on activation below.

To review clonal selection, try problem 13-4.

    B. T cells -- similar process as with B cells -- one type of protein with unique binding site made per cell & acts as trap. However, there are differences -- (See top of handout 24B for B vs T, and T_C vs T_H )

1. Protein made by T cell is T cell receptor, not Ab. (See Sadava fig. 42.11 (18.12)). Each T cell makes a unique TCR (also called T cell antigen receptor) due to DNA rearrangements of TCR genes. 

2. T cell receptor always remains on cell surface; never secreted

3. Antigen must be on eukaryotic cell surface:

a. Antibody will bind to free antigen in solution (or to part of a whole bacterium). TCR will not.

b. TCR only binds to Ag on surface of another (euk.) cell.  

c. T_C vs T_H

(1). T_C

(a). Target cell: T_C binds to ordinary (euk.) cell with abnormal epitopes on surface for example, an infected cell.

(b). Result of binding to target cell: T_C destroys target.

(c). Surface marker: T_C is CD8⁺ -- has protein called CD8 on surface

(2). T_H 

(a). Target cells: T_H binds to immune system cell with abnormal epitopes on surface, for example, a B cell that has bound Ag.

(b). Result of binding to target cell: Binding activates the T_H cell &/or B cell. (Promotes the immune response -- details vary depending on type of immune cell.)

(c). Surface marker: T_H is CD4⁺ -- has protein called CD4 on surface

    C. Clonal vs. Natural Selection. There are many similarities between clonal selection and natural selection. See section at end of lecture.

III. Activation of B and T cells -- what triggers clonal expansion?  See Sadava fig. 42. 13 (18.15) for overall picture.

  A. Why is activation needed?

1. For Clonal Expansion: a B or T cell must be activated in order to divide and specialize, and to form both memory cells and effector cells. See Sadava figs. 42.7 & 42.13 (18.6 & 18.15).

2. What do  effector cells do?

a. Effector B cells (plasma cells) secrete antibody

b. Effector T_C cells kill targets

c. Effector T_H cells  provide juxtacrine and paracrine signals that promote the activation and functioning of other immune cells.

    B. What is required? To activate a B or T cell, cell must get juxtacrine signals and paracrine signals. See Sadava table 42.4 & fig. 42.12 (fig. 18.14)

1. Paracrine = a cytokine = secreted protein that affects development of the immune system & some related functions.

a. Terminology: Cytokines made by leucocytes often called  interleukins, abbreviated IL-1, IL-2, etc.  

b. Example of action: Activation of both B and T_C cells requires paracrines from T_H cells. See texts if you are interested in names and functions of various cytokines. (No details of paracrines will be covered in class; some details are included here and in problem book FYI only.)

2. Juxtacrine --  Involves contact between surface proteins on two cells -- a T cell & its partner (target cell).  See handouts 24B & C and Sadava fig. 42.13 (18.14 or 18.15). At least two juxtacrine interactions are required:

a. T cell must have:  TCR  & CD4 or CD8: CD4 (if it's a T_H) or CD8 (if it's a T_C)

b. Partner must have epitope plus MHC on its surface. (MHC = cell surface protein; details below).

(1). TCR binds to epitope -- provides specificity of Ag/Ab or Ag/TCR match

(2). CD4 or CD8 binds to MHC -- type of MHC distinguishes targets to be 'helped' (by T_H) from those to be destroyed (by T_C ).

c. Other proteins are involved too; we are ignoring them. Consult advanced texts if you are interested.

    C. Role of MHC See Sadava fig. 42. 13 (18.15) for activation and role of MHC.

1. What is it? MHC = very variable surface protein. (MHC is an acronym for major histocompatibility complex.) Related in structure to antibodies and TCRs. (For pictures of MHC molecules see picture from Alberts two types of MHC)

2. What does MHC do? MHC and small pieces of antigen (epitopes or antigenic determinants) form a complex. Complex is on cell surface, so epitopes are 'displayed' on the cell surface, stuck to the MHC molecules.  

3. Two types of MHC

a. MHC I. All nucleated cells have MHC I on their surface.

b. MHC II. On cells of immune system only.

(1). Only certain cells of immune system (phagocytes & B cells) have MHC II on their surface.

(2). Not all T cells have MHC II at all times, & we will assume T cells do not have MHC II.

c. What proteins pair up?

(1). CD4 binds to MHC II

(2). CD8 binds to MHC I

d. What cells pair up? Two types of T's bind to different MHC's (w/ Ag) -- this is how T cells tell immune cells (that have captured Ag) and infected (ordinary) cells apart.

(1). Cytotoxic T's (CD8⁺) bind to target cells with Ag + MHC I on surface. 

(a). T_C are said to be "MHC I restricted" -- note target must have MHC I and Ag.

(b). Target cells for cytoxic T's are usually ordinary cells making abnormal proteins -- infected cells, for example.

(c). Binding to target (abnormal) cell → final activation of T_C and killing of target cell. (Additional earlier steps of activation are required; we are ignoring them.)

(2). Helper T's (CD4⁺) bind to  target cells with Ag + MHC II on surface.

(a). T_H are said to be "MHC II restricted" -- note target must have MHC II and Ag.

(b). Target cells for helper T's are usually cells of the immune system, especially B cells and phagocytic cells (macrophages & dendritic cells) that have internalized foreign antigens.

(c) APCs: Cells with pieces of foreign Ag on their surface (with MHC II) are called 'antigen presenting cells' or APC's.  For an example, see Sadava fig. 42. 12 (18.13)

(c). Binding to target cell (APC) → activation of T_H.

(d). Activated T_H can then activate a B cell or T_C. In some cases, if APC is a  B cell, activation can be mutual. (See 'one step' below.)

e. Activation of B cells  

(1). Two step:

(a). Phagocytic APC (not a B cell) binds to and activates a T_H 

(b).  Activated T_H detaches from APC; binds to and activates a B cell.  

(2). One step: B and T_H bind to and activate each other. B acts as APC to activate T_H; T_H  in turn activates B.

4. How does epitope get on MHC? How are antigens 'presented' or 'displayed' on the cell surface?

a. How the pieces get to attached to MHC -- depends on type of cell and where protein comes from.

(1). Infected cells -- proteins made inside the cell are digested in proteosomes; protein fragments (epitopes) enter the ER using a special transporter, and bind to MHC I.

(2). Cells of immune system -- proteins made outside the cell are engulfed (by phagocytic cells) or endocytosed (after binding to antibody on surface of B cells). Protein fragments bind MHC II in endosomes. See Sadava fig. 42.12 (18.13)

b. How MHC + epitope reaches cell surface --  MHC's  are transmembrane proteins in subcellular membranes (of endomembrane system); MHC's bind epitope and complex reaches cell surface by exocytosis.

c. Many epitopes displayed per cell -- every APC (antigen presenting cell) 'presents' many different pieces of whatever antigen(s) it engulfed, endocytosed or made. 

IV. How do T cells get activated? Summary Table. See also Sadava, table 42.2 (fig. 18.14) .

Notes:
(1). Activation of lymphocytes also requires appropriate  cytokines. T_H cells need IL-1 from the APC's; T_C cells need IL-2 from T_H and B cells need various IL's; class of Ab made by B cell depends on type of IL it gets.

(2) In a T_H -- B cell combo, each can activate the other. Alternatively, a helper T can be activated first, and then activate a B cell.

Try Problems 13-5, 13-9 & 13-13. For a review of the information so far, try 13-6 &, 13-11 (skip C).
 

V. How do cells make so many different antibodies, MHC molecules and TCRs?

    A. For MHC --  the answer is straightforward -- there are multiple genes for MHC, and each gene has many different alleles.

    B. How get so many different Antibodies & TCRs? Two major proposals:

1. Instructional theory: many antibodies have the same sequence, but fold differently around their respective antigens to take on different 3D shapes.

2. Selection theory: each antibody is different in sequence, and therefore has a different 3D shape to match a different antigen (epitope).

Q: Which idea fits best with what you know about protein structure and immune system function?

    C. If cells make different antibodies with different sequences, how to have enough genes?

1. DNA coding for antibodies & TCRs must be rearranged in each B or T cell.

2. You inherit a 'kit' of short DNA sequences and rearrange the parts to make functional genes for BCRs and TCRs. For details, see texts.

3.  We'll concentrate on antibodies, since the process is similar for TCRs.

4. To see how this works, we have to take a closer look at the structure of antibodies.

 VI. Ab Structure  -- See handout 24B bottom, picture of an immunoglobulin (from Alberts), or Sadava fig. 42.9 (18.9). What is the molecular structure of antibody molecules?

   A. V  vs C -- types of Immunoglobulin (Ig).  See handout 24B, picture of an immunoglobulin (from Alberts), or Sadava fig. 42.9 (18.9).

1. There are 5 main classes of Antibody -- IgM, IgD, IgG, IgE, and IgA. See table on handout 24B & Sadava Table 42.3 (18.3).

2. V & C: Each Ab or Ig is made up of a V section ("variable" region or Vee) & a C section ("constant" region or Cee).

3. Variable region

a. V is specific for Ag (or epitope). Determines what Ag will be bound = grabbers. 

 b. V is variable due to differences in sequence, not just differences in folding around Ag. 

c. Every Ab or Immunogloblin (Ig) has (at least) 2 grabbers.

d. All grabbers in one Ab are the same.

e. All the antibodies made by one Ab-producing cell have the same V. All the antibodies made by descendents of that cell have very similar V's.

Note: Minor differences are due to somatic mutation; see advanced texts if you are interested. We will ignore somatic mutation for the rest of this discussion, and assume all the antibodies made by the descendents of one cell have the same variable region.

f. DNA coding for V region is rearranged during development -- therefore a huge number of different V sequences are possible.

4. Constant region 

a. C determines biological effects -- localization of Ab, and what will happen as consequence of binding Ag. (Whether complement will be activated, whether Ab will be found primarily in blood or secretions, etc.)

b. 5 main types of C regions (in the DNA), therefore 5 main classes of antibody. (For properties of the dif. classes see handout 24B or Sadava Table 42.3 (18.3 )

c. The same V's can go with different C's.  (Called "Class Switching")

(1). All the antibodies made by one Ab-producing cell do not necessarily have the same C.

(2).The antibodies made by descendents of a single cell may have different C's. The same variable region can go with different constant regions as B cell clone expands.

(3). How can a different C go with the same V?

(a). By alternate processing/splicing of the RNA.

(b). By further rearrangement of the DNA to put a new C next to the V.  More below; for more details, see texts.

    B. H vs L.  See handout 24B or Sadava fig. 42.9(18.9)

1. Every Ig has 2 kinds of chains, L ("light") and H ("heavy"). Light and heavy refer to relative differences in mol. wt. 

2. Basic unit is 2 of each for a total of 4 chains. (For number of basic four-chain units per Ig, see table.)

3. Variable region (grabber) made of parts of each.

4. Each chain has a constant region -- Details FYI.

a. 2 kinds for L (kappa or lambda) 

b. 5 basic kinds for H (mu, delta, gamma, epsilon or alpha)

c. H_c (constant part of H) determines class (IgM, IgD, IgG, IgE, IgA)

d. Class (determined by H_c) determines location & other aspects of function (see "special properties" in table)

d. Class switching involves the H chains only, not the L chains.

To review structure of antibodies, try Problems 13-1 to 13-3.

5.   Myelomas & Hybridomas: Ig structure was figured out by studying proteins made by myeloma cells (cancers derived from Ab-producing cells) or hybridomas (hybrids of Ab-producing normal cells and cancer cells). Only way to get large numbers of cells all making the same Ab/Ig.  See texts for significance of hybridomas and monoclonal antibodies.  (Sadava fig. 42.10 (18.11))

VII. Structure of the DNA coding for Antibodies -- Basis of  Generation of Diversity (G.O.D) and Class Switching

    A. Basic idea: genes for H and L are mosaic -- Each "Gene" has several parts. See Sadava fig. 42.15 (18.16.)

    B.  How "gene" is divided  -- region coding for each chain (H or L) has parts coding for each type of constant region and several parts coding for the variable region .

    C. How DNA is used to make different antibodies (With different V's) -- DNA is rearranged

1. Pre Ag (See Sadava fig. 42.16 (18.170)

a. Rearrange short regions of DNA to make one coding region for variable part of H chain per naive/virgin B.  Delete all the intervening short segments.

b. A similar process of DNA rearrangement occurs in DNA coding for variable part of L chain.

c. Net: Only one H chain allele and one L chain allele are rearranged and used. Therefore each cell makes only one type of variable region.

2 Post Ag -- Alt splice of RNA and/or further rearrangement of DNA for H chain →  new mRNA →  new version of antibody with same variable region, but different 'constant'. Can have either of the following:

a. Rearrangement (usually deletion) of DNA → gene for H chain with original variable region and a new "constant" region. Make new class of antibody. (See Sadava fig. 42.17 (18.18))

b. Alternative splicing → mRNA for secreted version of cell surface antibody -- Same H chain except it's missing part that anchors the protein in the plasma membrane. Go from making "BCR" to making secreted antibody. This example discussed previously.

To review what happens before and after exposure to antigen, try problem 13-10 (skip D). Ig = immunoglobulin = antibody.

3. FYI: Post Ag -- Somatic Mutation** → minor changes in region of DNA coding for V regions of H  & L chains. (No change in DNA coding for C regions ). In the secondary response, there is a second round of clonal selection for B cell variants making 'better Ab' -- Ab that binds Ag better (higher affinity Ab). This is why Ab made in secondary response is better at binding Ag than primary Ab.

4. Reminder: Switching at DNA level is unique to immune system. Parts of genes for antibodies (or TCRs) can be rearranged using enzymes that cut DNA and join (recombine) DNA segments that were not contiguous in the germline DNA. These enzymes are restricted to certain immune cells.

**Note: We are going to ignore the effects of somatic mutation, but it is included here for reference.

    D. TCR genes are similar, except no somatic mutation. Genes are mosaic, and are rearranged. The proteins that are made have more than one chain; each TCR has constant and variable regions. See TCR picture from Alberts.

VIII. Evolutionary Aspects (FYI)

    A. Clonal vs. Natural Selection. Note how clonal selection and natural selection compare. In both cases, need to have many variants (diff. antibodies or dif. organisms) to be able to respond to unpredictable environmental challenges. How is this done? In both cases, make many variants and conditions select (promote propagation of) cells making the few suitable Ab (or carrying out a rare, useful function); the rest are wasted. Random generation of variants seems wasteful, but is the biological solution to preparing for change without conscious planning ahead.

    B. The Major Proteins of the Immune System are Related

1. The immune system uses 3 types of proteins that have a common evolutionary origin. These are antibodies, TCR and MHC.  For additional pictures see Sadava fig. 42.9 (fig. 18.9) for antibodies & Sadava fig. 42.11 (18.12) for TCR. Here are links to parallel pictures (from Alberts) of the two types of MHC, a TCR, and an immunoglobulin (showing the domains).

2. All 3 types of proteins have a "constant" part and a "variable part."

a. Constant part determines where protein is (cell surface? What kind of cell? etc.) and its general function.

b. All 3 proteins bind epitopes -- Variable part determines what antigen/epitope will bind to the protein.

3. All 3 proteins include one or more copies of the immunoglobulin domain -- a section of the protein that is similar in structure and function. this is a common theme --  the same domains are found over and over in different proteins. (Examples are SH2 domains; DNA binding domains, etc.)

4. Variable part of antibodies and TCR's are generated by rearranging the DNA; the variable part of MHC's is encoded in the germ line -- the DNA inherited in the zygote is the DNA used to code for the MHC's. The DNA from MHC is NOT rearranged. However the genes for MHC's are polymorphic (have many different common alleles).

IX.  Summary of Major Players in the Immune System:

The chart above summarizes the major players in immunology. You should be able to describe what each item is, its significance, and how it is related to all the others. "Secreted proteins" refers to those made by B and T cells. Proteins involved in the immune response (such as complement*) that are not made by lymphocytes are not listed. See ans. to problem 13-6, & tables above.

*Terms with a star have not been discussed in detail, but you should be aware of their general roles. You will not be asked any questions on the final about perforin, complement, somatic mutation, or which cytokine (or IL) does what.

Next Lecture (Optional) will cover regulation of the cell cycle, growth, and cancer.