Final ExamMolecular Immunology

Fall 2006

1. (8 pts) List as many ways as you can that the immune system can eliminate a bacterial infection.

Innate System: Neutrophils, macrophages direct kill by ROS, anti-bacterial peptides.

Adaptive Immune System: Ab + complement, ADCC, CTL killing of infected cells, T cells/CD1

2. (10 pts) Explain why many autoimmune diseases are associated with infections of particular pathogens. To get full credit you must explain two parts:

a. The infection partThere were a number of acceptable answers to this part of the question. Here is one: Infection leads to inflammation, which leads to more B7 production on APCs. This could result in self-antigen being presented by MHC and binding to TCR with costimulatory molecule also present.

b. The particular pathogen partSome pathogens may have peptides that are very similar to our own proteins. This could be adaptive for the pathogen since most TCR would have been negatively selected, but if a BCR bound to the pathogen’s protein and then through somatic hypermutation bound to a self-peptide better, this could explain why certain pathogens are associated with particular autoimmune diseases.

3. (6 pts) IL-3 and GM-CSF are both hematopoietin type cytokines, with similar receptors. They exhibit redundancy. Describe why the structure of their receptors makes redundancy an expected outcome

The cytokine receptors for these cytokines have a cytokine specific chain and a common chain. The chain is the signaling moiety and thus the two cytokines will transmit identical signals. This explains the redundancy observed between these cytokines.

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4. Mast cell degranulation is a complicated business. Describe: a. (4 pts) The trigger for degranulation IgE antibodies generated in response to an allergen bind to FcRI molecules on mast cells. Subsequent allergen exposure leads to crosslinking of the bound IgE and mast cell degranulation.

b. (5 pts) The role of Ca++ in degranulation, and how levels are changed.

The second messenger IP3 causes release of Ca from the ER. Activation of PMT leads to conversion of PE to PC. Accumulation of PC on the membrane surface increases membrane fluidity, which leads to an influx of external Ca. Ca is needed for microtubule assembly and fusion of the granules with the membrane (SNARE formation). Ca also works with PTK-activated MAPK to activate phospholipase A2, which promotes the breakdown of PC to lyso-PC and arachodonic acid.

c. (4 pts) How prostaglandins and leukotrienes are produced

Phospholipase A2 breaks down phosphatidylcholine (PC) to lyso-PC and arachodonic acid. Arachodonic acid is broken down into leukotrienes and prostaglandins by lipoxygenase and cyclooxygenase pathways, respectively.

5. (10 pts) Diversity in the adaptive immune system is found both within an individual and within the population as a whole. Describe the mechanisms that result in diversity in each situation.

Diversity in the population arises through polymorphism in the MHC, which leads to a large number of alleles for MHC class I, class II, TAP and proteosome components being expressed in the population. These molecules determine the ability of a given individual to mount an immune response against a given antigen. By having a large number of alleles in the population, the survival of the population is enhanced through this diversity. Diversity in the individual arises through the expression of Ig and TcR molecules within an individual. Ig and TcR molecules arise through non-homologous recombination of somatic gene segments. Each individual has a unique set of highly diverse Ig and TcR molecules that allow the individual to recognize >1011different antigens.

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6. (8 pts) IL-4 stimulates Th2 development via the STAT6 pathway; IL-12 stimulates Th1 development via the STAT4 pathway. In addition to the critical role of STAT proteins in Th cell development, transcription factors that act as “master switches” have also recently been described. Describe these factors and how they are thought to contribute to the stability of the differentiated Th cell populations.

IL-12 binding to IL12R activates STAT4, which leads to activation of the master transcription factor Tbet. Tbet in turns activates SOCS1, which suppress the activity of the IL4R and promotes Th1 development

IL-4 binding to IL4R activates STAT6, which leads to activation of the master transcription factor GATA3. GATA3 activates SOCS3, which suppresses that activity of the IL12 R and promotes Th2 development.

7. (16 pts) A major characteristic of the immune response is self-tolerance. Describe how self-tolerance is established in T and B cell populations. Be sure to state the components that are involved in the establishment of tolerance and where each form of tolerance you describe occurs.

Central tolerance: Occurs in the primary lymphoid organ, thymus for T cells and bone marrow for B cells. T cells expressing TcR specific for MHC:self Ag are eliminated during negative selection. B cells expressing Ig specific for self Ag are either eliminated during negative selection or undergo light chain editing.

Peripheral tolerance: Occurs in the periphery, often the lymph node. B cells that bind to self antigen are generally eliminated or anergized due to the lack of second signal from Th cells (CD40:CD40L). T cell tolerance is maintained via lack of second signal (CD28:B7), presence of Treg, presence of CTLA4, antigen sequestering.

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8. The effect of the MHC on the immune response to peptides of the influenza virus nucleoprotein was studied in H-2b mice that had been previously immunized with live influenza virus. The CTL activity of primed H-2b lymphocytes was determined by in vitro cytotoxicity assays (Chromium release) using H-2k fibroblasts transfected with H-2b genes as target cells. The target cells were infected with either live influenza or incubated with peptides from the influenza nucleoprotein. The results are shown in the table below.

System Target Cells (H-2k)

Test Ag CTL Activity

(% Lysis)A Untransfected Live influenza 0B Transfected w/ Db Live influenza 60C Transfected w/ Db Nucleoprotein

peptide 365-38050

D Transfected w/ Db Nucleoprotein peptide 50-63

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E Transfected w/ Kb Nucleoprotein peptide 365-380

0.5

F Transfected w/ Kb Nucleoprotein peptide 50-63

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a. (2 pts) Why was there no killing of the target cells in system A even though the target cells were infected with live influenza?

T cells are self restricted and the CTLs in this assay are of the H-2b haplotype, while the targets in group A are of the H-2k haplotype.

b. (4 pts) Why was a CTL response generated to the nucleoprotein in system C, even though it is an internal viral protein?

Because following infection, the virus is processed via the endogenous antigen-processing pathway and the resulting peptides are presented on class I molecules.

c. (5 pts) Suggest a reason why was there a good CTL response in System C to peptide 365-380, whereas there was no response in System D to peptide 50-63?Peptide 50-63 may lack a Db binding motif and thus wouldn’t bind to the MHC class I and be recognized by the specific T cell.

d. (5 pts) If you were going to develop a synthetic peptide vaccine for influenza in humans, how would these results obtained in mice influence your design of a vaccine?

You must be cognizant of the effect of MHC haplotype on the presentation of peptides, which determines the ability to mount an immune response. A peptide able to bind multiple haplotypes or a vaccine containing multiple peptides would be best.

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9. (10 pts) Cytokines regulate the immune system, mediating the quality, quantity and duration of the immune response, yet with a few exceptions, cytokines have not proven to have significant clinical value. Describe two potential reasons for this and why these characteristics lead to poor clinical efficacy.

Short half life requires injection of bolus amounts of cytokine, which alters the delicate balance of cytokine systems in vivo, resulting in expression of inhibitory cytokines or cytokine antagonists and thus limiting cytokine effectivness.

Cytokines exhibit plieotrophy, meaning that they act on multiple cell types and may have different effects on different cell types. Thus application of a cytokine may result in undesirable side effects.

10. (8 pts) Vertebrates express both adaptive and innate immunity. One theory is that the adaptive immune response evolved out of the innate immune response. This theory is supported by the existence of certain cell types, that appear to “bridge the gap” between the two systems, having greater specificity than innate cells, but less diversity than cells of the adaptive immune system. Describe a cell type that fits the category of a “primitive” adaptive immune cell.

Again there were a number of answers to this question. Here is one: NKT cells: these cells express markers of NK cells and T cells. They are TcR positive, but have a limited repertoire of T cell receptors, suggesting a cell that is in between evolutionary stages.

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11. You have developed two new anti-tumor therapies (Therapy A or Therapy B) that result in inhibition of tumor cell growth and eventual tumor elimination in a mouse model. To determine the mechanism behind your new therapies you perform the treatments in the presence of anti-MHC class I antibodies or in mice deficient for RAG2. The results are shown below, the Day 0 is the first day after therapy; a decrease in tumor volume indicates successful therapy.

a. (8 pts) In Therapy A, which cell type is likely acting as the effector cell, i.e. killing the tumor cell? Support your answer with the data presented in the graphs.

CD8+ T cell: Therapy A of tumor bearing RAG2 animals does not result in tumor growth inhibition, therefore cells of the adaptive immune response, B cells or T cells, are implied as the effector cell type. However efficacy is also eliminated when therapy A is used in conjunction with antibodies against class I, ruling out B cells and suggesting CD8 cells as the effector cell population.

b. (6 pts) How could you conclusively proof that the cell types you suggest is the one involved?

You could perform the experiments in mice depleted of CD8 cells or in 2 KO mice that had CD8 cells adoptively transferred in.

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0

100

200

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400

500

0 5 10 15 20 25

Time (days)

Tu

mo

r V

olu

me

No Treatment

Anti-MHC I

Therapy A

Therapy A + AntiMHC 1

RAG2 KO

c. (8 pts) In Therapy B, which cell type is likely acting as the effector cell? Again, support your data with the data presented in the graphs.

I MSTAKENLY ALTERED THIS QUESTION DURING THE EXAM, MAKING IT IMPOSSIBLE TO ANSWER. THEREFORE EVERYONE GOT FULL CREDIT FOR PARTS C AND D.

d. (5 pts) What potential explanation could account for the increase in tumor control by this cell type when anti-MHC class I antibody is added to Therapy B?

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0

100

200

300

400

500

0 5 10 15 20 25

Time (days)T

um

or

Vo

lum

e

No Treatment

Anti-MHC I

Therapy B

Therapy B + AntiMHC 1

RAG2 KO

Therapy B + AntiIL2Rbeta

12. (10 pts) Innate and adaptive immunity act in cooperative and interdependent ways to protect the host. Discuss the collaboration of these two forms of immunity. (NOTE: Although I have given you a whole page, you should confine your answer to 1-2 short paragraphs.)

In the case of pathogen infection, an innate cell such as a dendritic cell first recognizes the pathogen. Recognition occurs via the TLRs, which results in DC activation and cytokine secretion. Pathogen antigens are processed and presented by the DC in the context of MHC class II or class I molecules. In the case of class I this occurs via cross presentation. The presented antigens stimulate Th and CD8 T cells. The Th cells secrete cytokines that then act to stimulate the adaptive immune response and the innate immune response. In the case of the innate immune response the Th cell can trigger macrophage activation leading to increased phagocytosis.

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13. Answer each of the following questions concerning NK cells:a. (5 pt) NK cells kill their targets nonspecifically, but not randomly. Explain

the mechanisms that allow this to be true.

NK activity is controlled by the binding of activating and inhibitory receptors. When more activating receptors are engaged, the target is killed. Inhibitory receptors primarily bind MHC alleles and activating receptors recognize stress ligands. Thus NK cells do not kill randomly.

b. (5 pt) NK cells recognize their targets directly and indirectly. Give an example of an indirect and direct recognition mechanism.

Direct recognition involves recognition by activating receptors. Indirect recognition involves binding of Ig bound to the target cells by Fc receptors on the NK cell.

c. (5 pt) NK cells evaluate the level of MHC expression on potential target cells directly and indirectly. How does this occur? Be specific in terms of the proteins recognized and what recognizes them.

NK cells directly recognize MHC levels via inhibitory receptors and indirectly via recognition of HLA-E. HLA-E binds peptides from the leader region of HLA-A, B and C alleles. Thus in the absence of HLA-A, B and C there is no HLA-E.

d. (3 pt) NK cells differ from CTL in a number of ways. List two differences between NK cells and CTL. Give one similarity between NK cells and CTL.

There are phenotypic differences: NK cells lack CD3 and TcR, but express CD16 and CD56.There are activation differences: NK cells constitutively contain granules for killing; CTLs do not.Both cell types have similar killing mechanisms: perforin and granzyme release, induction of apoptosis.

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