1. (5) What organ is full of lymphocytes but does not support adaptive immune responses? What are such organs called?

Correct. I would be happy with "thymus" alone since I asked for "organ" (singular), which I did because the bone marrow is mostly things other than lymphocytes. 5/5

2. (22) List, in the first column of a table, the kinds of leukocytes found in blood which can be distinguished on a differential smear slide count with a light microscope with ordinary colored stains (without receptor-specific antibodies). Indicate whether each type of leukocyte is: a granulocyte, phagocytic, able to be clonally selected. Finally, tell very briefly what is the main function of each cell type. Your table header should look like this. Put the names of the cell type in alphabetical order.

Group 2's answers for the last column are in double quotes.

18/22 (4 points lost on functions (worth 2 points each): one each for lymphocytes and eosinophils; two for monocytes).

NK cells are lymphocytes and cannot be distinguished from B and T cells in an ordinary microscopic differential. So they could be mentioned under function of lymphocytes but I would not require it.

3. (10) What two kinds of cells are fused to make a hybridoma able to produce a monoclonal antibody? List the critical properties each parent cell must have.

Good answer, full credit! A minor error is that in fact plasma cells don't work when making antibody-producing hybridomas -- it is an earlier stage of B cell which must be fused. Optional information which could be included: The myeloma cell provides a plasma cell differentiated state conducive to production of large amounts of secreted antibody.

4. (8) In what organ, and in what stage of development, does what kind of leukocyte express both CD4 and CD8?

Good. If you wish, you could be a bit more precise about the end: 'ending after the cells complete alpha chain rearrangement and expression and survive both positive and negative selection.' 8/8

5. (12) What percentage of siblings of an F1 cross between two different inbred strains of mice will be MHC identical? What percentage of human siblings sharing the same father and mother will be MHC identical? Make some simple genetic diagrams which illustrate how to arrive at these answers, and explain briefly.

"Inbred mice are unique in the fact that at any given gene locus, they have only one allele for that gene. This means that they can only donate that one allele to their progeny. For two interbreeding inbred strains, this means that all of the F1 progeny will be identical genotypically for every locus. For MHC there is no difference. However, MHC is different from most genes, phenotypically, in that it demonstrates codominant expression which means that it expesses the allele received from both the mother and the father. Thus, all of the F1 progeny will be suitable donors to all of the other siblings.

• Mother: AA
• Father: BB
• F1: AB"

The above answer is correct, except that MHC is similar to most genes in having codominant expression. Remember, the only genes which don't show codominant expression (which means they show allelic exclusion) are Ig, TCR, and X-chromosome genes in females. All the other autosomal genes are codominantly expressed. 5/6

For humans, group 5 says [my additions in square brackets]:

For human situations we typically have a male and female parent with two different alleles for MHC [at each MHC locus]. Once again, however, we see codominant expression. This leads to four different possible genotypes for the F1, which means that there is a one in four or 25% chance that any given child will be able to donate to a sibling. Mother: AB Father: ab F1: Aa, Ab, aB, Bb"

Correct, 6/6.

Here is the way I wrote this answer, including the diagrams requested:

Cross between two different inbred mouse strains. Letters represent MHC allelic clusters on the two chromosomes of each mouse. Inbred mice are homozygous.

aa  X   bb
|       |
---------
    |
    ab

Cross between two humans who are likely heterozygous at most MHC loci.

  ab  X   cd
  |       |
  ---------
      |
-------------
|   |   |   |
ab  ac  ad  bd    

6. (30)

1. (6) List the six major HLA loci. "For class I: A, B, C For class II: DP, DQ, DR". Correct.
2. (2) How many alleles are you likely to have at each of these loci? "2 alleles". Correct (you as an individual, as I emphasized during the exam).
3. (6) Which cells in your body express each of these loci? "professional antigen presenting cells". Incomplete answer. 2/6. A good answer: 'All cells express class I (A, B, C). Class II (DP, DQ, DR) proteins are expressed by professional antigen presenting cells, or by most cell types in a zone of inflammation, induced by cytokines present during inflammation (notably gamma interferon).'
4. (4) What percent of your cells express HLA which you inherited from your mother? "all cells". Correct (all cells express class I, and it is codominantly expressed.) But not stated in the form requested, namely 100%. The form doesn't matter here, but it has mattered in the past, for example when I ask for a numeric answer and I get a numerator and denominator without a quotient stated. Please state the answer in the form requested. 4/4.
5. (3) Normally, how many HLA proteins of each class are expressed on one of the cells of your thyroid gland? "6 = class I MHC". Correct (3 loci A, B, C times two alleles/locus = 6).
6. (2) What circumstances would increase the number of kinds of HLA molecules expressed by that thyroid cell? (Explain briefly) "sustained inflammatory response, viral infection". Correct as far as it goes, but the "brief explanation" requested is missing. Interferon gamma and/or other cytokines would induce expression of class II MHC on the thyroid cells. 1/2
7. (7) Is it possible to express more class II HLA proteins than you have gene loci for class II? Explain briefly. "Yes, combinational assembly of the MHC chain". On the right track, but too vague. 'Chain' singular? Which MHC? 2/7. 'Yes, combinatorial assembly of MHC class II alpha and beta chains" would earn full credit. A thorough answer: 'Yes. Each locus (DP, DQ, DR) codes for at least one alpha and one beta chain. These form heterodimer molecules. Heterodimers form from chains within each locus (3 loci x 2 alleles = 6 proteins) but can also form from chains from different loci (up to 6 additional proteins, for a total of 12).'

7. (30) Describe the degree of "specificity" involved in binding of

1. TCR to antigenic peptide.
2. TCR to MHC.
3. MHC to antigenic peptide.

1. "TCR to Antigenic peptide is very specific, and each TCR recognizes only one specific peptide. The TCR variable domains are involved and the 2 CDR3 loops interact with the antigenic peptide. The generation of diversity of the CDR3 loops is mainly accomplished by Junctional flexibility, P-nucleotide addition and N-nucleotide addition." Good answer as far as it goes, but loses 4 points from failure to mention combinatorial diversity from V,D,J at CDR3. 6/10
2. "TCR to MHC is very specific, and each TCR recognizes only one specific MHC. The CDR1 and CDR2 loops of the TCR interact with the MHC. Generation of diversity of the CDR1 and CDR2 loops is accomplished by combinatorial joining of V-gene segments, however this diversity is rather limited compared to the diversity generated in the CDR3 loops." Excellent answer, 10/10. The answer seems to imply combinatorial diversity in CDR1/2, but in fact these are simply coded for by the V gene and not further diversified. Also, the limitation of a given TCR to "one peptide and one MHC" is a bit oversimplified, as we'll see when we get more into alloreactivity (graft rejection).
3. "MHC to antigenic peptide is NOT very specific. Each type of MHC molecule can bind a [[unique ??]] [very large] set of peptides. MHC molecule is referred to as being 'promiscuous'. Nonameric peptides with 9 [8-9] amino acid residues anchor to class I MHC, and longer amino acids of 13-18 residues bind to class II MHC. The diversity generated by the MHC molecule results from Polymorphism, that is the presence of multiple alleles at a given locus within a species. The diversity possible for a given species is an astronomical number [,] 10 to [the] 12th. This creates a major obstacle when it comes to matching MHC molecules for succesful organ transplants." Basically a good answer. 10/10. The 'astronomical' sentence is a bit unclear: diversity at the entire MHC region is spoken of, not diversity at a single locus. Here is the answer I wrote, which is shorter yet complete: 'Little specificity. Each MHC can bind thousands of different peptides, though there are certain sequence restrictions at the 'anchor residue' positions in the peptide. The diversity comes from the pool of MHC alleles in the population.'

8. (43) B lymphocytes go through a complex, multistep process during maturation, which results in each naive but immunocompetent cell expressing a unique immunoglobulin receptor for antigen.

1. (2) What does the abbreviation CDR stand for? "CDR: complementarity determining region". Correct.
2. (4) What is the function of the CDR's in the immunoglobulin protein? "CDRs constitute the antigen binding site. They determine the diversity and the specificity of antibodies." Good, correct.
3. (3) Where do the CDR3's lie in the epitope-binding surface. "CDRs are in the center of the antigen binding surface." Correct.
4. (6) What must a B cell do in order to receive help from a T cell? "The B cell must bind the antigen [with its immunoglobulin antigen-receptor, internalize it via recepter-mediated endocytosis], process the antigen, and express it in a complex with MHC class II on its membrane. The B cell must also express CD40 which will interact with CD40L which is present on the T helper cell [and B7]." 5/6, one point off for B7.
5. (6) What does help from a T cell to a B cell consist of? "Completion of the competemce [competence] signal which leads to the expression of receptors specific for cytokines released from the T helper cell. These cytokines will cause the B cell to proliferate and differentiate." 4/6, 2 points off for omitting signalling via costimulatory receptors such as MHC II and B7.
6. (6) What changes in the structure of the immunoglobulin made by a B cell follow, and require, help from a T cell? "Class switching is induced by specific cytokines released by T helper cells. Class switching involves changes in the Fc portion of the immunoglobulin giving rise to a new class, but [the resulting Ig] has the same variable regions for antigen specific interactions." 3/6, points off for omitting induction of somatic mutation.
7. (10) Can a B cell synthesize two isotypes of antibody at the same time? Explain briefly. "Yes. After RNA processing a naive B cell will express mIgM and mIgD." Hmmm. Not what I hand in mind, but absolutely correct. I should have said 'secrete two at the same time'. My answer was: 'No. Isotype switching requires the DNA recombination of the V(D)J gene with a constant region gene. This process cuts out and discards the intervening constant region gene(s), and is irreversible. Once it occurs, the B cell can synthesize only the new isotype.' So I'll accept either answer or a combination. 10/10.
8. (6) Can a B cell synthesize both integral-membrane Ig and secreted Ig at the same time? Explain briefly. "No. Naive B cells produce only membrane-bound antibody and plasma [[B]] cells produce only secreted antibody. Membrane boung [bound] and secreted antibodies to the same antigen only [[have difference]][differ] at the c-terminal [C terminus] of their heavy chains. This difference comes from the differential processing of a common primary transcript, which cannot occur at the same time in the same cell." Actually the answer is yes (which I stated both in lecture and in review session). A small portion of the mRNA is processed differently from the majority. But this answer shows understanding of the main point, control via mRNA processing. 5/6.

End of questions for Part I.

Questions for parts II-IV.

9. (15) A monoclonal IgG to CD45 has an association equilibrium constant of ten to the eighth (10⁸) liters per mole. A suspension of lymphocytes is incubated in a solution containing 0.15 micrograms/milliliter of the antibody. At equilibrium, will at least half of the CD45 receptors on the cells be bound with antibody? (Assume that the amount of antibody bound by the cells is small enough that it has no effect on the free antibody concentration in solution. This assumption is usually valid under typical experimental conditions.) Show your calculations and briefly explain how you arrived at your answer.

The calculations are as follows:

1. Ka = 1.0 * 10^8 L/mol = [Ag:Ab]/[Ag][Ab]
   Kd = 1.0 *10^-8 mol/L = [Ag][Ab]/[Ab:Ag]

2. [Ab] = 0.15 micrograms/mL
   (0.15ug/mL)(1000mL/L)(1mg/1000ug)(1g/1000mg)(1mol/150,000g) = 1.0 * 10^-9 mol/L
   [Ab] = 1.0 *10^-9 mol/L

3. [Ab] < Kd 1.0 * 10^-9 mol/L < 1.0 * 10^-8 mol/L"

Correct, full credit!

I've included another presentation of the same answer which might help some people see how to think this question thru on their own:

1. Half saturation occurs when the free antibody concentration is equal to Kd. Kd = 1/Ka, so Kd = 10^-8 molar (moles/liter).

2. We need antibody in moles/liter, which in turn needs grams/liter, so:
   • First, convert micrograms of antibody to grams:
     (0.15 micrograms)(10^-6 grams/microgram) = 0.15 x 10^-6 grams.

   • Now, convert grams of antibody to moles (The molecular weight of IgG is 150,000):
     (0.15 x 10^-6 grams)(1/150,000 moles/gram) = 1 x 10^-12 moles.

   • Now, convert milliliters to liters:
     (1 milliliter)(10^-3 liters/milliliter) = 10^-3 liters.

   • Finally, divide moles by liters to get molar concentration of antibody:
     (1 x 10^-12 moles)/(10^-3 liters) = 1 x 10^-9 molar.

3. This is one tenth of the concentration needed to half-saturate the CD45 receptors on the cells, so the answer is NO.

10. (35) Mice injected with trinitrophenol (TNP) make no immune response. Mice injected with hapten-conjugated TNP-chicken gamma globulin (TNP-CGG) make antibody specific for the hapten (and also for the CGG). The TNP-specific antibody binds TNP, or TNP conjugated to any protein, regardless of the protein, since the antibody in question is specific only for the hapten TNP.

(a). It was found that mouse B memory cells primed with TNP-CGG, but specific for hapten TNP, could receive help from syngeneic mouse Th memory cells specific for unrelated unconjugated bovine serum albumin (BSA). However this worked only when the B + T cells were provided with TNP-conjugated BSA. This help enabled the B cells to make anti-TNP antibody. This experiment was done in culture, and only the B and T cells were present. Explain step by step what the B cell, and the T cell, are doing in this situation.

"The TNP-conjugated BSA is recognized by the TNP[-]specific B [no hyphen] cell. It is internalized by endocytosis and degraded into peptides (peptides containin [containing] BSA [you mean to say peptides derived from BSA]). [I'd like to see you mention low pH and lysosomal enzymes.] These BSA peptides are associated with the class II MHC on the B-cell and are recognized by the T(helper)-cell (which is BSA specific). The B/T-cell conjugate is formed and the T-cell releases cytokines that induce the B-cell's activation. [Th also help via costimulatory receptors.] The B-cell proliferates and differentiates to eventually produce multiple B-cells that have antibodies specific for TNP hapten."

Good answer, full credit!

(b). A culture was set up containing a mixture of TNP-specific B memory cells with BSA-specific Th memory cells. To this was added a mixture of TNP-CGG and unconjugated BSA. This culture produced no anti-TNP antibody response. Explain which steps occurred, and which failed, in this case. Note that two cell types mentioned above are the only cell types put into the experiment.

Good answer, full credit! (You don't need to hyphenate B cells and T cells, though.)

11. (30/30) When a virus enters body fluids, but before it infects cells, antibody provides effective immunity by rendering the virus particles noninfectious. However, once a virus has infected cells in the body, cytotoxic T lymphocytes are required to eliminate the infection. Using molecular biology techniques, it is possible to clone the gene for a virus protein, express it as a protein, and purify the protein. (Human insulin, a protein hormone produced in this way, is now in wide use by insulin-dependent diabetics.) (For your answer, accept the previous statements as facts which need not be questioned.) Will a molecularly-cloned virus protein make the best possible vaccine for a situation in which some viruses will manage to infect cells, despite the presence of antibody? If yes, explain in detail. If no, explain in detail, suggest an approach which would be more effective, and tell why. (No information from Chapter 18, Vaccines, is necessary to answer this question fully.)

Good, but leaves out 'the antigen needs to enter the cytoplasm, and be processed by the proteosome, transported to the endoplasmic reticulum with the transporters of antigenic peptides (TAP), and presented in class I MHC'. 12/15

"A more effective approach would be the use of an attenuated viral vaccine. The attenuated virus is an inactivated virus that enters the cell and is processed endogenously. The processed peptides are expressed on class I MHC molecules and presented on the cell surface membrane of the infected cell. Cytotoxic T lymphocytes will recognize the viral peptides and subsequently proliferate and differentiate into effector and memory T cytotoxic lymphocytes. Thus, subsequent viral infection will elicit a heightened immune response along with the elimination of the virus."

Good answer, but the word 'inactivated' is misused, and I can't tell whether the writers understand that infection and virus gene expression is needed. An inactivated virus is not capable of infecting a cell, hence cannot direct the endogenous synthesis of viral proteins. The term needed is attenuated, which means the virus can still infect cells but is not virulent (does not cause disease). 12/15

Additional information of interest but not needed in the answer: Vaccinia (cowpox) is an avirulent relative of the smallpox virus; rabies was the first virus attenuated in the lab. Recently, virus genes have been introduced into avirulent viruses such as vaccinia and found to induce CD8 CTL. Another emerging vaccination method uses injection of naked viral DNA as the vaccine. The DNA does not include the entire virus genome and produces no infection. However it gets into cells enough to be expressed as virus proteins and produce an immune response. Finally, protein vaccines can be introduced into cell cytoplasm by encapsulation in ISCOMS (see Fig. 18-5, p. 454), or other adjuvant methods.

12. (20/20) You are working for a biotech company. A colleague proposes that the company should make a clone of human T cells specific for an antigen found in a high percentage of human breast cancers. Some of the cloned cells could then be injected into breast cancer patients, after the conventional course of surgery, chemotherapy, and/or radiation is completed. The goal would be to kill any remaining cancer cells. As the company's immunologist, you are asked to evaluate the feasability of this plan. (Assume that the breast cancer antigen information is correct; concentrate on the immunology.)

Recognition will fail due to restriction by the MHC. The clone of T cells would be specific not only for the cancer epitope, but also for the MHC allele which presented that epitope. This is called restriction of T cell antigen recognition by MHC. Due to the large number of MHC alleles at any locus in the population, nearly all cancer patients will happen to have different alleles, and this clone will recognize the cancer epitope poorly in nearly all patients.

Rejection of injected T cells. The second serious problem is that because the T cell clone will express different MHC class I antigens than the recipient patient, the patient's immune system will reject the T cell clone, rendering it ineffective.

In conclusion, this idea is not feasable. [This is why Steve Rosenberg at the NIH has gone to the extreme of removing lymphocytes from each cancer patient, then activating and expanding them in culture, and then reinjecting them into the patient.]

Group 12 wrote this. Portions in [[...]] should be omitted. Other corrections are in [...]. I've added the boldface summary points (and it will help all of us if you include such summary points in any long answer you write!).

[[The feasability of]] using a clone of human t-cells [T cells, no hyphen, see below] wich [which] are specifc for human breast cancer gene products is not very feasable. The theory [idea, see below] is a good one, because the t-cells would be specfic to the breast cancer gene product. Also being able to inject a human t-cell into the patient would not cause an immune response because there would be no phylogenic distance between the patient and the cancer fighting t-cells.

There are many [[negative]] drawbacks to this theory. Must avoid graft-vs.-host reaction. The main problem is that the t-cell tcr [T-cell receptor, hyphen is correct, see below; or TCR because acronyms should be all upper case] must not react with the hosts MHC as an autoimmune disorder. The tcr must be able to bind with the host cells [cells'] MHC class one molecules and not concider [consider] the host [host's] normal cells as foriegn [foreign] or non self. There are several hundred different alleles in the human HLA gene loci. Therefore the biotech company must produce a t-cell which will bind to each of these different HLA gene products. Restriction of recognition. Also the tcr of the t-cell must be able to bind to the breast cancer gene products which are presented on the host's cells MHC class one molecule. The biotech company will have to produce a t-cell which will have a high affininity for the breast cancer gene product and be able to interact with the hosts MHC class one molecules and not treat them as foriegn. This process will cost a great deal of money and time, but the benefits are also high. Now there will be a t-cell which could be injected into the patient and kill the cancerous cells and can interact with every HLA genotype."

On a take-home answer, I expect good grammar, spelling, and capitalization. Isn't it distracting to read an answer with so many easily avoidable errors?

Group 12's answer points out the problem with restriction of recognition by MHC, although this phrase is not used. Good. But it fails to recognize one of the major problems: rejection of the T cell clone by the patient.

The possibility of a graft vs. host reaction is real (4 point gain), though not one of the two major problems.

Bottom line: 10 + 4 - 2 (poor presentation) = 12/20.

A point of terminology: A theory is a hypothesis which is accepted as correct by the majority of scientists based on experimental evidence. Therefore this is a 'proposal' or an 'idea', not a theory.

Another point of terminology concerning when to hyphenate: The terms T cell or T lymphocytes should not be hyphenated unless they modify another term. This is the same rule used for all such hyphenation. For example, it is correct to describe a woman who comes in from the cold as having a red face, but if we use "red face" to modify another word, it should be hyphenated ("red-faced woman"). The hypen shows that "red" modifies "faced", not "woman". Similarly , it is correct to say that T cells express the T-cell receptor ("T" modifies "cell", not "receptor"), and to say that cytotoxic T lymphocytes, which are a T-cell subpopulation, carry out CTL-mediated killing. ("CTL" modifies "mediated", not "killing". On the other hand, "cytotoxic" modifies "lymphocytes", not "T"; hence there is no hyphen.)