Biology 432 Name _________KEY______________

Introduction to Immunology

Exam 2

100 points possible

 

        Before beginning, please verify that you have 8 pages..

        Please read each question carefully before answering.

 

 

Short Answer (86 points)

 

1. (16 points) Compare and contrast the Pre-BCR and Pre-TCR proteins by completing the following table:

 

Topic to Address	Pre- B Cell Receptor (Pre-BCR)	Pre-T Cell Receptor (Pre-TCR)
Cellular expression (stage of lymphocyte maturation when these receptors are expressed)	Pre B Cell   (Or one could say that it is the stage when the heavy chain of membrane bound IgM is expressed with surrogate light chain and Iga/Igb, see below)	Pre T cell   (Or one could say that it is the stage when VbDbJb is complete and TCRb chain is now expressed in a double negative Pre T cell, see below)
Major protein components	Surrogate light chain=VpreB, and l5 IgM heavy chain Ig a/Igb	Pre T a Pre TCR TCR b CD3
Two signals supplied to maturing lymphocytes by activated receptors	1. Stops V to DHJH arrangement of heavy chain (may be responsible for allelic exclusion)     2. Induces light chain rearrangement	Only 2 of 4 possible responses were required: 1. Cell becomes permissive for TCRa chain locus arrangement 2.     Stimulated expression of CD4 and CD8 co -receptors 3.     Stimulates proliferation 4.     Stops additional TCR b chain locus arrangement

2.     (14 points) Using the illustration above, answer the following questions:

a.     To initiate the generation of mature T cells, lymphoid precursors migrate to the thymus to generate a population of dividing cells or thymocytes. How does the expression of c-kit aid in the process described above?

         C-kit expression and binding to SCF is important for the survival of thymocytes migrating to and initially within the cortex of the thymus

 

b.                       If thymic stromal cells were eliminated from the thymus which region of the thymus would be altered and which process of selection would be eliminated?

 

Thymic stromal cells are nonhematopoietic cells. This question is asking about the role of epithelial cells in the cortex. Epithelial cells in the cortex, expressing antigen on class I or class II MHC, are important for positive selection of immature thymocytes or double positive ProT cells.

 

Short Answer: CORTEX, POSITIVE SELECTION

 

c.                       What mechanism is used by Pro-T cells to ensure that more than one combination of an abTCR is present with a clone of cells that will undergo positive selection? Why is this mechanism important?

 

Combinational joining. Combinational joining increased the diversity of the TCR and the probability of generating receptors with optimum affinity for MHC+petide. Answers addressing the rearrangement of the TCR b chain, proliferation of the PreT a chain, followed by rearrangement of a mature a chain to yield a diverse TCR population were also accepted.

 

d.         Where within the thymus does negative selection take place and what is the purpose of negative selection?

            MEDULLA.

The purpose of negative selection is to generate self-tolerant T cells expressing TCR, and either CD4 or CD8.

 

e.          Given what you know about negative selection and the avidity hypothesis, indicate how the process of T cell selection might change in a mutant mouse whose medullary cells could not activate ERK (also known as MAPK).

 

A large, transient increase in activated ERK kinase is thought to provide the strong signal associated with apoptosis of self-reactive T cells during negative selection. Without activated ERK kinase this transient increase would not take place and there would be no negative selection. The presence of autoreactive T cells could be present in the mutant mouse.

(Also, one could discuss the role of ERK in positive selection. Without a sustained, low-level ERK kinase signal positive selection would not occur and developing T cells would be eliminated before negative selection.)

 

3.     (8 points) Monoclonal antibody to CD3 activates a powerful proliferative response in T helper cells in vitro as long as adherent dendritic cells and activated macrophages are present in the in vitro cell cultures. If dendritic cells and activated macrophages are removed, the proliferation of T helper cells decreases. Explain these results. (Hint: Indicate which signal(s) would be missing in cultures without dendritic cells and activated macrophages.)

This monoclonal antibody binds to CD3 and activates the intracellular signaling machinery downstream of the TCR receptor. In effect the T cell will respond to antibody binding as if the TCR has been engaged by MHC+peptide (Signal 1). However, the results show that full activation of the T cells requires interaction with dendritic cells and activated macrophages. These cells are antigen presenting cells that not only express MHC+ peptide but also the co-stimulatory protein B7. B7 must bind to CD28 in order to provide a complete signal to activate the T cell and stimulate proliferation. Removal of the dendritic cells and activated macrophages removes the co-stimulatory B7 signal.

 

 

Name the co-stimulatory T helper protein that, if expressed and activated would mimick the effects of removing adherent cells from these cultures as described above.

 

                  CTLA-4

 

 

4.     (10 points) T cells respond to genetic differences in allografts by recognizing alloantigens. What are alloantigens?

                               Antigenic determinants present on molecules that differ among members of the same species because of genetic variation.

 

 

Alloantigens can be recognized by the host’s T cells following direct or indirect presentation by APC’s. Describe the difference between direct and indirect presentation.

                               

Direct: host T cells recognize MHC+peptide on the surface of cells of an allograft

 

Indirect: host T cells recognize MHC+peptide on the surface of self antigen presenting cells that have endocytosed or phagocytosed antigen expressed by an allograft

 

 

How does cross-reactivity of TCR-antigen interactions and negative selection of T cells during maturation contribute to the presence of alloreactive T cells?

                                     

The ability of a T cell to bind to a foreign MHC molecule is due exclusively to crossreactivity. The T cells of the host that originally are selected to recognize foreign peptides+self-MHC and survive negative selection, cross react with portions of foreign MHC molecules that are structurally identical to foreign-peptide-self-MHC combinations that the T cells normally recognize.

 

 

5.     (12 points) Generation of high affinity antibodies takes place within the germinal centers of lymph nodes. Imagine that you have generated a mouse strain whose follicular dendritic cells (FDC’s) )are devoid of membrane-bound proteins. Indicate whether you would expect evaluation of your mouse model to provide the following results by writing “Yes” or “No” on the line preceding the statement. Provide a one or two sentence explanation defending your answer for each statement.

 

a.     __Yes______In the germinal centers of the lymph node, the mutant mouse strain is likely to show no changes in centroblast number and somatic mutation rates.

 

It is likely that these mice will show no changes in centroblast number and somatic mutation rates sine proliferation and somatic mutation take place in the dark zone before contact with FDC’s.

 

 

b.     ____No____ Alteration in surface proteins expressed by FDC’s should not influence cellular events in germinal centers since FDC’s do not normally express receptors important for B cell differentiation and antibody production.

 

FDC’s express Fc receptors and complement receptors which are used to display antigen to centrocytes. Antigen-antibody complexes are formed during centrocyte binding and are used to select for centrocytes with high-affintiy binding immunoglobulin and to provide a survival signal. Without this interaction witn FDC’s and their receptors, centrocytes undergo apoptosis.

 

 

c.     ___No_____ The light zone of the germinal center is likely to increase in size since centrocytes with both high and low affinity immunoglobulin should proliferate.

 

Again, without interaction with FDC’s centrocytes should undergo apoptosis. There should be a loss of centrocytes in the light zone with this mutation. The size of the light zone should decrease as number of both high and low affinity centrocytes dwindle.

 

 

d.     ____No____ Class switching and maturation of memory and plasma cells expressing and secreting high affinity antibodies should not change since T helper cells will function normally in this mutant mouse model.

 

Without FDC’s survival signals, centrocytes will not survive through the light zone to reach T helper cells.

 

Yes, could be accepted if the answer was explained similar to the following: If however, there were some centrocytes to move past FDC’s contact-mediated selection, they could interact with T helper cells. However, whether they would secrete high or low affinity antibodies is not clear with the removal of FDC’s and the selection process for high affinity immunoglobulin.

6.     (10 points)                    

Mouse Strain (DBA/2 x A) F₁ (H-2 ^d/a)

 

Maternal MHC

 

 

 

 

                                          Paternal MHC

 

 

 

 

Given the maternal and paternal haplotypes of the MHC complex shown above, answer the following questions regarding T cell activation by antigen presenting cells or target cells expressing proteins encoded by the MHC complex shown above.

 

 

 

Which antigen-presenting MHC proteins on the surface of an activated B cell isolated from the F₁ mouse will be recognized by a mature T cell (TCR) isolated from an H-2^a strain?

 

                        Class II MHC are used for antigen presentation.

                        IAa^a b^a and IEa^a b^a class II MHC proteins will be recognized.

 

 

 

Following activation of the H-2^a T cell, which cytokine will be produced by the T cell to stimulate clonal expansion and generation of memory and effector T cells?

                                       

                                                IL-2

 

 

 

 

Would a CTL from a H-2^k/a strain recognize and respond to a virally infected self-cells in the F₁ mouse? Defend your answer with a brief explanation (one or two sentences).

 

 

                                    Yes. K^a, D^a, and L^a class I MHC proteins would be recognized.

 

7. Fill-in-the-Blank (14 points, 2 points each)

Please place the term(s) on the line that is best described by the definition to its right.

 

A. tolerance (anergy accepted)     a state of unresponsiveness to antigen in both B and T cells produced by prior exposure to that antigen

 

 

B. E2A (BSAP accepted)             B cell transcription factor regulating the expression of RAG proteins and progression through the pre B cell stage

 

 

C. b₂ microglobulin           immunoglobulin protein whose interaction with peptide and class

(calnexin also accepted)   I a chain is essential for proper folding of class I MHC

 

 

D. dendritic cell                 phagocytic antigen presenting cell that constitutively expresses class II MHC and B7

 

 

E. alpha 1 and alpha2        both regions of class I MHC responsible for binding processed antigen for presentation

                                                                                                        

 

F. CD4                              monomeric TCR co-receptor with four extracellular Ig-like regions

 

 

G. IL-7                              cytokine that is secreted by bone marrow stromal cells and bound by Pre-B cells during maturation

 

 

 

7.         (16 points) B cell activation by a thymus-dependent antigen requires an initial signal

(signal 1) mediated by antigen cross-linking followed by a second signal provided by cell-cell interaction with T helper cells.

 

Provide the correct terms in the paragraphs discussing activation of the BCR by a TD antigen.

 

 

 

Mature B cells first recognize a TD antigen with the ____VARIABLE CDR______ regions of their surface BCR. After cross-linking, signal transduction pathways are activated and the antigen-antibody complex is internalized through receptor- mediated endocytosis.

 

Cross-linking a surface antibody, leads first to the activation of ____SRC_______ kinases which create docking sites for ____SYK________ kinase (a B cell specific kinase) by phosphorylating ____TYROSINE________ (amino acid) in the __ITAM________ regions of the cytoplamic tail of the Iga/Igb complex. Phosphorylation of ____SYK________ kinase (B cell kinase listed above that is similar to the ______ZAP-70_______ kinase in T cells), stimulates a cascade of events resulting in the activation of PLCg and ______GEF’s__________ which activate small GTP binding proteins.

 

PLCg hydrolyzes PIP2 in the membrane generating two second messengers ___DAG______ and ______IP3____. These intracellular messengers activate PKC and calcium mediated pathways required for activation of gene transcription and other functional changes in the activated B cell.

 

After internalization of the antigen-antibody complex by the B cell, internalized antigen does through the ______ENDOCYTIC__________ processing pathway and is presented by ____CLASS II_________ MHC. In order for presentation of peptide+ MHC, expression of the non-classical MHC protein ______HLA-DM________ is required to exchange CLIP for the TD antigenic peptide.

 

T helper cells bind the antigen-MHC complex with TCR and its co-receptor CD4. Other co-stimulatory proteins such as _____B7_______ on the B cell and CD28 on the T cell provide additional signals to both T and B cells. Signal 2 is provided to the B cell by the upregulation of ___CD40L______ on the T cell and its interaction with ____CD40_______ on the B cell.

 

Completion of signal 2 stimulates the expression of cytokine receptors on the B cell allowing the B cell to respond to cytokines being secreted from the activated T cell. B cells begin to proliferate and differentiate into memory and plasma B cells.

 

 

 

Bonus (10 points)

1. (4 points) In what way does linkage disequilibrium explain the reality of the diversity of MHC expression in an outbred population.

 

The theoretical diversity of MHC expression in an outbred population is estimated to be 2.25 X10 18 possible combinations of class I and class II alleles. Theorectical diversity assumes that the assembly and expression of class I and class II proteins is completely random. Actual diversity of MHC proteins is less because certain allelic combinations occur more frequently thanwould be predicted by random combination. Linkage disequilibrium is the difference between the theoretical or expected diversity and the actual diversity of MHC molecules. Reasons for linkage disequilibrium vary but include the possibility that some MHC molecules are “more fit” than others or that too few generations have gone by for all possible crossover events to take place and yield expected diversity.

 

 

 

2. (4 points) Notch receptor interaction with Notch ligands has recently been proposed to regulate Th1 and Th2 responses in T cells. Indicate which Notch ligands stimulate either Th1 of Th2 responses.

 

Delta-Notch binding is thought to stimulate a Th1 response.

 

                              Jagged-Notch binding is thought to stimulate a Th2 response.

 

 

 

3. (2 points) Name the molecule used to process antigen in TAP-deficient cells and that is recognized by gdT cells and NK cells.

 

CD1