BIO 432                                                                      Name________KEY____

Introduction to Immunology

Exam 3

 

        Before beginning, please verify that you have 7 pages.

        Please read each question carefully before answering.

 

I.      Essay/Long Answer (54 points)

 

A. (28 points) CD4+ and CD8+ T-cells leave the thymus and enter circulation in resting G0 state.  Recirculation of T cells between the blood and lymph occurs every 12 to 24 hours until they encounter antigen or undergo apoptosis.  Describe the process of T cell activation, proliferation and differentiation in a lymph node by answering the following questions:

 

1.  What region of the lymph node is rich in T cells and is the most likely site of CD4+ T cell activation?  Which antigen presenting cell(s) is most likely to present circulating antigen to T cells in this region of the lymph node?

 

 

Paracortex

                        Macrophages and Interdigitating dendritic cells

 

 

  1. CD4+ or CD8+ T lymphoblasts differentiate into one of three possible effector T  cell types.  What are the three possible subpopulations of effector T cells generated during differentiation?  Describe the function of the differentiated effector T cell subpopulations.

 

The answers below are the minimum required information for credit:

 

TH1-- classic cell mediated adaptive immune response, activation of macrophages and production of delayed type hypersensitivity, activation of CTLs, secrete cytokines such as IL-2, IFN-g, IFN-b

 

TH2—activate B cells, stimulate class switch to mIgE, activation of eosinophils

 

CTL-lysis of viral and latered self-cells, secrete perforin and granzymes

 

( Another possible answer: Peripheral gd T cells and their function)

 

 

 

 

 

 

  1.  Essay/Long Answer (54 points)

 

  1. CD8+ effector T cells are distinguished by the expression of specific cytokines, enzymes, and cell adhesion molecules.    What are the signals that are required to activate CD8+ effector T cells?  Following activation, what soluble effector molecules are produced by CD8+ effector T cells to mediate target cell destruction.

 

    1. antigen –specific signal transmitted by TCR complex-peptide class I MHC
    2. co-stimulatory signal mediated by the binding of CD28-B7
    3. binding of IL-2 to the IL-2 receptors

Soluble effector molecules or CTLs= perforin, granzymes, IFNg

 

 

 

 

  1. Explain why effector T cells have lower activation requirements than naïve T  cells.

 

Effector T cells express the membrane bound signaling molecule CD45RO.  Naïve T cells express the membrane bound signaling molecule CD45RA.  CD45RO readily associates with the T CR-CD3 co-receptor and activates Src kinases that phosphorylate ITAM tyrsoines on CD3 without CD28-B7 interaction and signal transduction.

Additonally, effector T cells increased their expression of cell adhesion molecules (CAM) 2 to 4 times that of naïve T cells.  Increased CAM expression results in stronger interactions between effector T cells and APC’s.

 

 

 

B. (26 points) B cell development is divided into two phases, an antigen-independent phase and an antigen dependent phase.  Answer the following questions regarding B cell maturation, activation and differentiation.

 

  1. B cell maturation takes place in the bone marrow and does not require the presence of antigen. Sequential expression of membrane bound immunoglobulin and Ig-a/Ig-b during maturation occurs as lymphocyte progenitors interact with cells in the bone marrow.  Describe the cellular interactions directing the maturation of immature B cells in the bone marrow.   Include in your answer the cell types and cell adhesion molecules involved in the interaction.  Indicate the pattern of expression of immunoglobulin and Ig-a/Ig-b at different stages of B cell differentiation in the bone marrow

The first step in B cell maturation is the interaction between lymphoid progenitors and stromal bone marrow cells.  VLA-4 expressed on lymphoid progenitors binds to vascular CAM-1 on bone marrow stromal cells.  Following this interaction, lymphoid progenitors expressing c-kit being to mature into pro B cells.   The pro B cells surface CAM c-kit interacts with SCF on stromal cells.  Binding of c-kit to SCF stimulates signal transduction pathways that upregulate the expression of the IL-7 receptor on pro B cells.  As stromal cells begin to synthesize IL-7, the IL-7 is bound by the IL-7R on pro B cells.  Intracellular signaling activated by the binding of IL-7 to the IL-7R leads to the maturation of pro B cells into pre B cells. Pre B cells do not require adhesion to stromal bone marrow cells for survival.  Pre B cells detach and migrate away from the bone marrow stromal cells and mature into immature B cells.

 

NOTE: YOUR TEXT DOES NOT DIFFERENTIATE BETWEEN LYMPHOID PROGENITORS AND PRO B CELLS.  I MAY HAVE TAKEN OFF POINTS FOR AN ANSWER THAT DID NOT DIFFERENTIATE BETWEEN LYMPHOID PROGENITOR CELLS AND PRO B CELLS.  CHECK YOUR EXAM , IF THIS IS THE CASE I WILL REPLACE THOSE POINTS.

 

                        Pattern of expression of Ig-a/Ig-b and BCR:

 

Ig-a/Ig-b is expressed at early pro B cell stage and throughout the rest of the maturation process.

 

Lymphoid progenitors and pro B cells do not express any form of the BCR on their membrane.  Pro B cells begin to synthesize and rearrange heavy and surrogate light chain genes.  Heavy chain rearrangement is completed in the pre B cell stage and Pre BCRs (heavy chain and the surrogate light chain) are expressed by pre B cells.  Light chain rearrangement begins and immature B cells express the mature BCR (heavy  chain and kappa or lambda light chains).

 

  1. Antigen-dependent activation, clonal deletion, and differentiation of B cells occurs outside of the bone marrow.   Activation requires the cross-linking of the BCR complex by antigen binding.   List the two classes of antigens that can cross-link the BCR receptor complex.  Describe the chemical nature of these two classes of antigens (ie. Provide an example of each type of antigen).  Discuss the two signals required for B cell activation by these classes of antigens.  Indicate whether these classes of antigen activate the BCR complex and stimulate signal transduction pathways that inhibit or enhance the activity of immature and mature B cells.

 

 

The two classes of antigens that can directly cross-link the BCR receptor complex are TI-1 and TI-2 antigens.  TD antigens do not cross-link the BCR effectively and require T helper cell activity for activation of B cells.  Competence and progressive signals are required for B cell activation.   Competence requires antigen recognition, cross-linkage of the BCR and CD3 activation and moves the B cells from G0 into a G1 stage.  Progression requires cytokine signaling and/or additional interactions with the BCR. Progression moves the B cell from G1 to S phase.

 

 

TI-1: bacterial cell wall components such as LPS; activate immature and mature B cells

           

           

TI-2: polymeric proteins, bacterial cell wall polysaccharides with several repeating units; activate mature B cells and inactivate immature B cells

           

 

 

  1. BCR activation can be modified by a co-receptor.  List the three proteins comprising the BCR co-receptor.  Provide one function of the co-receptor.

 

 

 

CD19, CD21, TAPA-1

This receptor amplifies BCR signaling allowing lower affinity mIgM to respond to low antigen concentration.

 

 

 

 

 

 

 

 

 

 

 

 

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

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

 

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

 

B. AICD                              homeostatic mechanism of Fas-FasL induced apoptosis important in removing T cells that repeatedly recognize antigen

 

C. E2A                                 B cell transcription factor regulating the expression of RAG1 and progression through the pre B cell stage

 

D. Elastase                           microbial component that inactivates anaphylatoxins C3a and C5a

 

E. Procaspase8                     inactive enzyme recruited to FADD following Fas-FasL binding

 

F. pleiotropy                       property of a cytokine that has different biological effects on different target cells

 

G. C1 inhibitor                     enzyme that regulates activation of the classical complement pathway by preventing excessive C2 and  C4 activation by C1q.

 

III.      Short Answer (32 points)

 

A.         (8 points) The humoral response generates high affinity antibodies with antigen specificity and particular effector functions.  Class switching is one of two cellular processes that takes place as antigen-stimulated B cells migrate through a germinal center to generate the humoral response.  Describe the process of class switching in a thymus-dependent antigen stimulated humoral response. Where in the germinal center does this process take place?  Which cell-cell signaling event is required for induction of class switching in centrocytes?  How do the cytokines, IFN-gamma or IL-4 influence the outcome (Ig expression) of class switching in differentiating centrocytes.

 

Class switching occurs in the light zone of a germinal center.

Class switch allows any given VH domain to associate with the constant region of any isotype.  Class switch maintains antigen specificity while allowing change in effector function.  CD40 on B cells must interact with CD40L on T helper cells.

INF gamma promotes class switching to IgG2a or IgG3.

IL-4 promotes class switching to IgE also IgG1.

 

 

 

B.        (8 points)  Identification of class I and class II cytokine receptors and several ligands for these receptors (eg. IL-4 and IFN-gamma) has lead to the development of a unifying intracellular signaling model for cytokines.  As cytokine binds the a subunit of the cytokine receptor while the b subunit initiates the signal transduction cascade.  Clearly describe the role of JAK and STAT proteins in the signal transduction event initiated by the activated b subunit.

 

 

Cytokine binds and dimerizes the cytokine receptor.  Dimerization induces the activation of JAK which autophosphorylates itself and tyrosines on the  b subunit of the cytokine receptor.  These phosphorylated tyrosines serve as docking sites for STAT proteins which recognize the phosphorylated tyrosines with SH2 domains.  Followingrecruitment and  binding of STATs, JAKs phosphorylate STATs.  Phosphorylated STATS dimerize and traslocate to the nucleus where their act and transcription factors and regulate gene transciption.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C.                 (16 points) The human immune system possesses three complement pathways, the classical pathway, the alternative pathway and the lectin pathway, that function to eliminate foreign antigen.  List the four primary functional roles of all three of these complement pathways.

 

                  1. lysis

2. opsonization

3. degranulation

4.              clearance of immune complexes

5.              activation of inflammation also accepted

 

Part 5. Below are three incomplete descriptions of the complement pathways.  Provide the appropriate term in each blank (0.5 point each).

 

Activation of the classical complement pathway begins Fc portion of membrane bound._IgM_or ___IgG_    plus antigen is recognized by  the complement protein _C1q .  Binding of two pairs of monomers, _C1s  and C1r  (each having catalytic domains and enzymatic activity) generates the first active enzyme of the complement pathway, C1qr2s2.

 

This activated enzyme cleaves C4 into C4a and C4b and C2 into C2a and C2b forming C4b2a, the active C3 convertase.  C3 convertase now cleaves hundreds of molecules of C3 into C3a and C3b. Some molecules of _C3b_ bind to the C3 convertase, to form ._C4b2a3b_, a C5 convertase that cleaves C5 into C5a and C5b. C5b binds to the surface of the target cell and subsequently binds C6, C7, C8, and a number of monomers of C9 to form C5b6789n, also known as the MAC.

 

 

Part 6. Generation of C3 convertase is a common amplification step in all three complement pathways.  Discuss how activation of the alternative and lectin pathways and the generation of active C3 convertase differ from the activation and generation of C3 convertase in the classical pathway described above. 

 

            The alternative pathway is initiated by spontaneous hydrolysis of C3. C3b binds Facto B exposing a site on Factor B that is cleaved by Factor D.  This generates C3bBb, the C3 convertase that is stabilized by properdin.

 

The lectin pathway is activated by the recognition mannose residues by mannose-binding lectin (MBL) .  MBL is bound by MASP-1 and MASP-2 which gerneate an aacitvated C1 like complex that can cleave C3 to generate the C3 convertase along the classical pathway.

 

 

 

 

 

IV.      Bonus  (8 points)

 

A.   (3 points) Which three proteins form the active apoptosome?

 

 

Cytochrome C, caspase 9, apaf-1

 

 

 

B.    (2 points)   Paroxymal nocturnal hemoglobinuria (PNH) is a disorder caused by a defect in the synthesis of two regulators of complement.  Name the two regulators of the complement pathway whose decreased expression results in PNH. 

 

 

DAF and MIRL

 

 

 

C.  (3 points) Innocent-bystander lysis is a potential side-effect following activation of the complement system.  Name the complement protein responsible for initiating the process of innocent-bystander lysis and one mechanism of regulating the non-specific activity of this complement protein.

 

 

            C5b67 and S protein

 

            (also HRF and MIRL)