C2006/F2402 '05-- Outline on Immunology -- revised 5/6/05 (typo found in III-A-5)

(c) 2005 Dr. Deborah Mowshowitz Columbia University, New York, NY. Last Update: 05/06/05 02:23 PM . Order of problems does not match order of topics below. It will probably be easier to go over the lecture first, and then try the problems.

Major Players in the Immune System:

Cells B cells, TC cells, TH cells, phagocytic cells, APC's
Secreted Proteins Antibodies (Ab or immunoglobulins; 5 classes), Perforin, Cytokines (Interleukins & interferons)
Cell Surface Proteins MHC, BCR, TCR, CD4, CD8

The chart above summarizes the major players in immunology. By the end of the this lecture, 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.

Handout: 25A (Antigen Presenting Cells & Activation of T cells) -- Posted version is from a previous year; there are some minor differences. Other handouts not on web.
 

I. What do antibodies and/or T cells do?  See the table at the end of the last lecture for major features of two branches of the immune system.

    A. Action of B cells to combat infection:

B cells --> release antibody --> Ab (antibody) binds Ag (antigen -- usually on surface of microbe) --> trigger destruction of microbes (microbes are engulfed by phagocytes or lysed -- see Purves 18.11) often with the help of complement.
Allergies are a side effect of this system.

    B. Action of  cytotoxic T cells

All T cells --> TCR on surface; TCR's of cytotoxic T's bind to Ag on surface of virus infected eukaryotic cell --> destroy target cell by triggering apoptosis. Cytotoxic T cells can trigger apoptosis by juxtacrine signaling; alternatively they can 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. (See Purves 18.14 (19.15)) This is why grafts fail; foreign cells of graft look like infected (defective?) cells and are destroyed. 

Note: 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.

    C. Action of helper T cells

All T cells --> TCR on surface; TCR's of helper T's bind to Ag on surface of cells of immune system. (Called APC's or antigen presenting cells.) Interaction helps activate one or both partners. More details on activation below.

    D. Cytokines. T cells do secrete something (in addition to perforins) -- called cytokines or lymphokines.

    E. Two major types of T cells -- how does each one do its respective function?

1. How do you tell the two types apart?  Surface proteins/markers on T cells and their significance. See handout 25B.

a. TH have the protein named CD4 on their surface (therefore are said to be CD4+)

(1). CD4 helps normal action of  TH  -- helps TH bind to normal target cell of immune system & helps activation of immune cell.

(2). CD4 serves as identifying marker for helper T's.

(3). HIV binds to CD4. Therefore CD4 (accidentally) acts as an HIV receptor (there are other co-receptors) -- allows HIV to enter helper T cells. HIV infection --> loss of helper T's --> complete loss of immune function. (See Purves 18.21 & 22)

b. TC have CD8 (are CD8+)

(1). CD8 helps normal function of TC -- helps TC bind to usual target cell = infected or rogue cell.

 (2). CD8  serves as identifying marker for cytotoxic T's.

2. (FYI only): More than one type of helper T's exist. Currently thought to be two major kinds -- TH1 (mostly helps macrophages and cytotoxic T's) and TH2 (helps B's to function). Details are beyond scope of this course, but this is currently a hot research area.

3. How do two types of T's match up with proper targets?

a. CD8 or CD4 binds to respective protein on surface of target cell.

b. CD8 on cytotoxic T binds to a protein -- MHC I -- found on surface of all nucleated eukaryotic cells.

c. CD4 on helper T binds to a different protein -- MHC II -- on surface of cells of immune system. (More details on MHC below.)

d. How do cytotoxic T's tell normal from infected cells? Presence of antigen plus MHC I.

II. Immune System -- Important Features to explain -- Review

    A. Specificity & Diversity

    B. Memory

    C. Tolerance

    D. Response is adaptable

(Also need to explain roles of all cells and proteins listed at start of lecture.)

III. Clonal Selection -- How do you account for the "important features" listed above? See handout 24.

    A. B cells (See Purves fig. 18.7 (19.7))

1. Each cell differentiates --> produces a single type of Ab on surface ("virgin" or "naive" B). Each cell rearranges its DNA during differentiation, so each cell has a unique set of Ab coding genes and makes a unique antibody -- that is, with a unique set of "grabbers."

Note: As B cells mature and specialize, changes in the antibody they make may occur because of alternative splicing and/or additional rearrangements of the DNA. Structure & rearrangement of Ab coding genes and antibodies is discussed below.

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

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 -- 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 (embryonic vs adult) and additional factors. (This turns out to be very complicated, so we are ignoring 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. (This is the selection part that accounts for specificity, diversity, and adaptability.)

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. (This is the clonal expansion part that accounts for memory & tolerance -- memory when Ag triggers multiplication, and tolerance when Ag triggers destruction or suppression).

5. Activation: Why do you need helper T cells? B cells must be activated for clonal expansion to occur (as in step 3b above.) Some antigens can activate B cells by binding to Ab (BCR) on the B cell surface. Most antigens can not activate B cells without the assistance of helper T cells. In most cases, the B cells must bind to antigen and to the surface of helper T cells in order to be activated.

    B. T cells -- Similar process as with B cells

1. DNA rearrangement occurs during development -- therefore one type of protein with unique binding site made per cell.

2. Protein made by T cell is T cell receptor, not Ab. (See Purves fig. 18.13(19.14)). Each T cell makes a unique TCR (also called T cell antigen receptor) due to DNA rearrangements of TCR genes. 

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

4. Clonal expansion occurs in response to Ag ---> more T cells -- effector cells & memory cells.

5. Activation: T cells require activation, as do B cells. T cell activation requires "antigen presentation." Antigen must be on surface of another cell (on an infected cell to activate a TC or on an APC to activate a TH).

IV. Activation: More on Antigen Presentation & Major Histocompatibility Complex (MHC).  (For pictures see Purves chap. 18).  

    A. What is MHC?

1. MHC = very variable surface protein. There are 2 main types, and many  versions of each type.  Each individual has several different genes for each of the two main types of MHC. Each of these genes has  20-40 or even more variants  (alleles). Since there are several genes per person and many different alleles of each gene in the population, there is a lot of variation in the actual MHC  proteins (and DNA) from person to person. These genes, unlike genes for antibodies and TCR's, do not rearrange during development. So there is variation from person to person, but all cells in a single person have the same MHC genes. 

2. Two types of MHC 

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

b. Cells of immune system (all APC's) have MHC II on their surface. (Not all T cells have MHC II at all times, & we will assume T cells do not have MHC II.)

    B. What are Antigen Presenting Cells (APC's)? APC's = Cells that have antigens bound to MHC  on their plasma membranes. How they get their antigens/epitopes and attach them to MHC is shown on the top of 25A. T cells bind to the MHC-Antigen complex, as shown in the middle of the handout. (See Purves 18.17 (19.17))

 1. APC's do not present whole antigens -- APC's present fragments of antigens called epitopes or antigenic determinants. See top 1/2 of 25A & See Purves 18.15 (19.16)

a. Ordinary cells (not from immune system) present fragments of whatever proteins they are making (+ MHC I). These epitopes come from proteins made inside the APC itself and then partially digested in proteosomes. (These cells are not "classic" APC's but serve a parallel function and will be called "APC's" in these notes.)

b. Immune system cells (B cells, dendritic cells & macrophages = "classic" APC's) present fragments of whatever they have engulfed or endocytosed (+ MHC II) -- Purves 18.16 (19.16). These epitopes come from proteins that were originally outside the APC and were partially digested in lysosomes/endosomes. 

2. Each APC presents many different epitopes at once (even if they are all derived from a single antigen).

3. How do the epitopes reach the cell surface?

a. The endogenous fragments digested in proteosomes enter the ER (by a special transporter), and combine with newly made MHC molecules (in the ER membrane). The complex is transported to the cell surface through the ER, Golgi, etc. in the same way as any cell surface protein. 

b. The exogenous fragments digested in lysosomes/endosomes combine with newly made MHC in the lysosomes/endosomes and the complex reaches the cell surface the same way that used receptors recycle to the surface.

    C. Some Terminology  (FYI)

1. T cells are said to be "MHC restricted;" B cells are not

a. B cells recognize plain Ag = Antibodies can bind to Ag in plasma or on bacterial/viral surfaces.

b. T cells recognize only Ag that is bound to MHC on (euk.) cell surface ( Purves 18.16 (19.17)).

(1). T cell receptors bind to variable part of MHC-Ag complex = bind to Ag itself

(2). CD4 or CD8 binds to constant part of corresponding MHC.

2. Two types of T's recognize (bind to) Ag associated with different MHC's -- this is how T cells tell immune cells and infected (ordinary) cells apart.

a. Cytotoxic T's (CD8+) recognize Ag + MHC I (said to be "MHC I restricted") -- note target must have MHC I and Ag.

b. Helper T's (CD4+) recognize Ag + MHC II (said to be "MHC II restricted") -- note target must have MHC II and Ag.

The point: T cells recognize their targets (in part) by the type of MHC they have -- infected cells have MHC I and immune cells have MHC II.

V. Putting it all together -- Purves 18.17 (19.18) or handout 25A

    A. T cell is activated (Middle of 25A)

1. T cell  & APC must match up surface proteins & Ag in order to bind.

a. APC must have Ag (epitope) + MHC

b. T cell must have TCR that matches Ag, and  CD4 or CD8 to match proper MHC.

Note: Picture on handout shows epitope in middle, in between both MHC of APC and TCR of T cell. The epitope is firmly bound to the MHC and stays with the APC when the T cell finishes activation and detaches. The activated T cell now has an empty TCR and will bind to another (B) cell with the same epitope.

2. Right type of APC must go with right type of T cell -- either

a. For helper T: Binding to classic APC (B cell or phagocytic cell -- macrophage or dendritic cell) needed to activate TH

(1). In primary response, APC probably a phagocytic cell (not specific for any particular antigen)

(2). In secondary Response, APC likely to be a B cell (with antibody specific for that antigen)

b. For cytotoxic T: Binding to infected cell needed to activate TC.

3. Cytokines must be provided for activation -- to bind to receptors on T cell. 

a. Cytokine (IL-1) from APC needed to activate TH .

b. Different cytokine (IL-2) from TH needed to activate TC. ( This is why you need TH's for cytotoxic T response.)

4. Activation --> clonal expansion (more T cells) AND more specialization of T cells. These activated T cells can disassociate from the APC and find another cell to "help" or kill.

    B. What Activated TC cell does (see bottom of handout 25A): Divides and/or kills an infected cell.

    C. What activated TH cell does (see bottom of handout 25A)

1. Humoral Response: Activated TH cell then divides and/or activates a B cell -- activates the same APC that just activated it (if it was a B cell) or finds a new B cell.

2. Cell Mediated Response: Activated TH cell divides and/or helps activate a TC cell (by providing cytokines) -- details of this not discussed. 

VI. How do T and B cells get activated? Summary. See handout 25A. and topic V above.

What's Activated?**

Antigen Presenting/Target Cell

What holds Epitope

Source of Antigen

Result 

Cytotoxic T

Infected Cell

MHCI 

Made in infect. cell

Killing of Target Cell;
Mitosis of TC cell --> clone

Helper T

Classic APC (B, macrophage, etc.). 

MHCII

From outside APC

Humoral Response as in (1) or (2) below; Mitosis of TH cell to give clone

 

  (1) macrophage

    MHCII

From outside by   Phagocytosis

Activated TH can go on to activate a B; leads to Ab production by B cell

 

  (2) B

    MHCII

From outside by   RME

Mutual Activation of TH and B;  Ab production by B cell

**Note: Activation of lymphocytes also requires appropriate  cytokines. TH cells need IL-1 from the APC's; TC cells need IL-2 from TH and B cells need various IL's; class of Ab made by B cell depends on type of IL it gets.

VII. Ab Structure -- How is specificity achieved? (Handout 25C or Purves 18.10 (19.11). This topic will probably be abbreviated in lecture, but is included here in detail to make it easier to follow.

    A. V  vs C -- types of Immunoglobulin (Ig).  

1. There are 5 main classes of Antibody -- IgM, IgD, IgG, IgE, and IgA. See table on handout 25C & Purves Table 18.3 (19.2).

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

3. Variable region

a. V is specific for Ag. 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. (Minor differences are due to somatic mutation; see 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.)

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, therefore 5 main classes of antibody. (For properties of the dif. classes see handout 25C or Purves Table 18.3 (19.2) )

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. How is this possible? Need a closer look at Ig structure

    B. H vs L.  See handout 25C or Purves 18.10 (19.11)

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

a. 2 kinds for L (kappa or lambda) 

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

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

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

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

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.  (Purves 18.12 (19.13))

    C. Classes of Ab and class switching during development of immune response

1. Order of events (see handout 25C) during immune response

a. First make M, then M + D -- all on surface.

b. Meet Ag --> primary response: secrete M.

c. Meet Ag a second time --> secondary response; secretes usually G but can be E or A.

d. All these Ig's combine with same Ag

2. Implications of structure and switching

a. Can make different variable regions -- zillions of them, one for each dif. epitope. So the IgG, for example, in a person is a mixture -- all IgG molecules have the same constant regions but have different variable regions.

b. During differ. stages of immune response, can make Ab with same variable region but different constant region (for H). Can switch class and/or secreted vs. surface. How is this possible?

3. What we already know: How switch from membrane bound to secreted works. Variable part stays same; Hc changes from hydrophobic to hydrophilic by alt. splicing/poly A addition.

4. What we don't know so far: How do you make so many dif. variable regions AND What changes when you switch classes (from IgM to IgG? M to M + D)?  Must be rearrangement of DNA or alternate splicing of RNA. Different solutions at different steps.

VIII. Structure of the DNA -- Basis of Class switching and generation of diversity (G.O.D)

    A. Basic idea: genes for H and L are mosaic -- Each "Gene" has several parts. See texts or handout or Purves 18.18 (19.19.)

    B. Switching occurs at DNA and RNA levels. (Switching at DNA level is unique to immune system.)

    C.  How "gene" is divided  -- region coding for each chain has parts for each type of constant region and several parts for variable region.

    D. How DNA is used to make different versions of the same antibody

1. Pre Ag -- rearrange V/D/J region of DNA to make one variable region per naive/virgin B. Alternate splicing allows cell to produce M and D antibodies with same variable region (but different constant region of H chain).  For details see Purves 18-19 (19-20).

2. Post Ag -- Alt splice of RNA and/or further rearrangement of DNA  --> new mRNA --> new version of antibody. Can have either of the following:

a. Rearrangement of DNA --> H chain with original variable region and a new "constant" region. Make new class of antibody. (See Purves 18.20 (19.21))

b. Alternative splicing --> 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.

    C. Summarize G.O.D -- how get so many V's?

1. H & L mix and match

2. V parts (V, D, J) mix and match

3. Joins are inexact (when you rearrange the DNA -- when join V to D etc.)

4. Somatic mutation (post Ag) --> minor changes in V, not C

    D. TCR is similar, except no somatic mutation

    E. 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.

IX. So how do other cells get differentiated (specialized) to carry out their particular functions?

Cells of the immune system specialize (differentiate) by rearranging their DNA. Cells of the rest of the body specialize in a different way  -- they keep the same DNA but they still make different proteins.  How do cells with the same DNA differentiate to make different proteins? If there is time we will discuss this briefly. If you want to know more, take a look at "Lecture 26" of 2004. This is provided FYI only -- the material in "lecture 26" will not be on the final.