Week 4

Question 1

• Describe how exogenous and endogenous antigens are processed.

Answer 1

o T cells cannot interact with native antigen. The antigen must first be processed and presented to T cells while bound to MHC molecules on the surface of APCs
• CD4+ T cells → MHC class II (Endolytic Pathway; exogenous Ag)
• CD8+ T cells → MHC class I (Cytosolic Pathway; endogenous Ag)
o Most cells in the body can present antigen using MHC class I molecules (both foreign and self-antigens) whereas professional APCs present antigen using MHC class II molecules
o APCs:
• DCs are able to participate in cross-presentation of exogenous antigen as it can express both MHC I and II molecules to activate CD4 and CD8 T cells
CD4 T cells secrete IL-2 which activates CD8 T cells

Question 2

• Describe how antigens are presented to the immune system on MHC Class I molecules.

Answer 2

o Cytosolic Proteolytic Pathway: endogenous peptides
• Average half-life of cellular proteins = 2 days; some are degraded within 10 minutes
• Protein degradation occurs through 20S proteasome in conjunction with 19S regulator → 26S proteasome
The constitutive proteasome can give way to the immunoproteasome when the cell is stimulated with IFNγ or TNFα as it changes out different subunits. The immunoproteasome more efficiently loads peptides in MHC class I molecules through better association with TAP protein complex
• With the immunoproteasome it will present proteins on MHC I molecules after degradation occurs
The Ubiquitin tagged protein binds to the 19S regulator and allows the proteasome to open and the protein to enter the barrel of the proteasome for degradation
• Peptides from this pathway are transported to the RER where they bind to a transporter protein heterodimer complex (TAP1 and TAP2). This complex extends across the RER membrane and facilitates passage of these peptides to the lumen of the RER where loading of the peptides to Class I molecules can take place
TAP = Transporter-associated with Antigen Processing
TAP has an affinity for peptides of 8-16 amino acids; optimal binding size = 9 amino acids
• Final trimming is accomplished in the ER by ERAAP (ER-associated aminopeptidase)
• Free amino acids remain in the cytosol
• MHC I molecules are made in the RER and associate with calnexin to ensure proper folding. Once folded properly it associates with the β2-microglobulin and calnexin is released
Several proteins associate with MHC I now:
• Tps (tapasin) allows the MHC I association with TAP1/2 complex
• ERAAP trims the protein into the appropriate size peptide as it travels to the CRT/p57 (precursor MHC I) molecule
• Degradation and presentation of peptides on Class I permits the sampling of proteins that are synthesized within the cell
DRiPs (defective ribosomal products) are rapidly degraded and presented on the cell surface associated with MHC Class I molecules
Allows for the recognition and killing of cells that have aberrant DNA → aberrant proteins
• Note, virus infected cells contain distinct 20S proteasome which is induced by IFNγ and TNFα and degrades and presents viral proteins on the cell surface via MHC I
Allows for the recognition and killing of cells that are infected with viruses

Question 3

• Describe how antigens are presented to the immune system on MHC Class II molecules.

Answer 3

o Endolytic Degradation Pathway: exogenous antigen
• Exogenous antigens are internalized into APCs through endocytosis or phagocytosis
DCs and macrophages tend to phagocytize antigens
B cells use receptor mediated endocytosis (BCRs or TLRs)
• Internalized antigens are degraded in phagolysosomes or endosomes
• Protein trafficking takes 1-3 hours for antigen to be processed and presented within the endocytic pathway
• MHC II molecules are synthesized in the RER as trimers (α and β chains coupled with an invariant molecule)
• The invariant molecule:
Assists in folding the Class II α and β chains
Binds to the peptide-presenting site of the Class II molecules
Assists in transport of MHC Class II molecules from Golgi to cytoplasmic vesicles
• Proteolytic cleavage gradually digests the invariant chain into a short fragment (CLIP) which is bound to the antigen presenting site on the MHC II molecule
CLIP = Class II-associated Invariant Chain Peptide
• A non-classical Class II MHC molecule is required to catalyze the exchange of antigenic peptide for CLIP: HLA-DM
HLA-DM can be regulated by HLA-DO. These are involved in regulating CLIP removal and peptide binding to MHC II
HLA-DO is only expressed in B cells and in the thymus; blocks HLA-DM except in very acidic conditions (late endosome).
• HLA-DO = HLA-DOA, or DNA, or DZA

Question 4

• Describe the costimulatory molecules essential for T cell activation.

Answer 4

page 7

Question 5

o MHC Class I Deficiency:

Answer 5

failure to process antigen
• Signs: severe bronchiectasis (dilation of bronchi from repeated infections), persistent cough, chronically ill, recurrent infections of sinuses, middle ears & lungs
• Etiology:
Deficiency of CD8 Killer T Cells (although labs can reveal that lymphocytes are normal, the differential of T cells shows a predominance of T helper cells and a deficiency of CD8 Killer T cells)
Reduced amounts of MHC class I molecules – not due to transcription levels but because the TAP 1 & 2 proteins are not functioning properly. Thus the peptide fragments are not getting into the RER and MHC cannot bind the peptide to present it on the cell surface
• Remember:
T cells need two instructional signals to become activated (effector) T cells
• Signals = Fas & granzymes/perforin
Once activated they need to recognize antigen in MHC I on target cells
• Despite profound deficiency in CD8 T cells and a severely decreased ability to present viral antigens to CD8 T cells, affected individuals were able to fight some viral infections since not all CD8 T cells were affected (able to present some)
• However, due to recurrent viral infections and the increased damage they caused resulted in bronchiectasis which results in increased susceptibility to bacterial infections
• Reduction of CD8 T cells is a direct result of lack of MHC Class I production
Thymocytes interact directly with MHC I which is crucial for maturation of CD8 T cells
• These individuals have overactive NK cells which cause granulomatous lesions on the skin of the face

Question 6

o MHC Class II Deficiency:

Answer 6

failure of gene regulation
• Onset = usually around 6 months of age, as maternal immunity is lost at this time
• Presents as a mild form of SCID (severe combined immunodeficiency)
SCID = no T cells are made and they do not respond to mitogen
MHC II Def. = reduced number of T cells and they mildly respond to mitogen
• Signs: increased susceptibility to pyogenic and opportunistic infections
Typically, MHC II deficient individuals also have moderate to severe hypogammaglobinemia (T cells are unable to activate humoral immunity)
• Labs:
Peripheral blood cells are able to be stimulated by mitogen (phytohemagglutinin – PHA) which tells T cells to proliferate → tests T cell function
Serum immunoglobulins are very low ☹ thus T cells are not able to activate the humoral arm of the immune system (B cells are not activated); however, allogenic B cells (B cells from another source) are able to present MHC II → issue with T cell population
High neutrophil count, low lymphocyte count (B cell fraction higher than normal)
• T-cell ratio = 1:3 for CD4:CD8 (Normal = 2:1, CD4:CD8)
• Thus CD4 T cell count is reduced
Flow Cytometry: decreased expression of HLA-DQ and HLA-DR molecules
• Etiology: autosomal recessive trait, but is NOT linked to the MHC class II gene found on chromosome 6; defect lies in regulation of expression of MHC II genes (defective transcription factors)
IFNγ normally induces expression of MHC II molecules on APCs; however, in individuals with MHC II Deficiency IFNγ does NOT induce MHC II formation

Question 7

• Be able to describe, in general terms, the process of B cell development and activation.

Answer 7

o B cell development occurs in the bone marrow and does not require contact with antigen
o The earliest stage of antigen-independent B cell development is the progenitor B cell (pro-B cell) stage and can be divided into three sub-stages based on TdT and B220 expression:
• Early pro B cell: TdT
• Intermediate pro-B cell: TdT and B220 (glycosylated state of CD45R)
• Late pro-B cell: B220, downregulated TdT
Note, B220 remains expressed on the B cell surface throughout the remainder of maturation
• As pro-B cells progress through this stage, they rearrange their Ig heavy chain genes and express CD43 (leukosialin, an adhesion factor to bind bone marrow stromal cells), CD19 (BCR co-receptor, works with CD21 and CD81), RAG-1 and RAG-2
• Pro-B cells also express c-Kit which binds to stem-cell factor expressed on bone marrow stromal cells which induces the pro-B cell to proliferate and differentiate into pre-B cells
o As late pro-B cells pass into the pre-B cell stage, they downregulate TdT, RAG-1 & 2, and CD43
o Pre-B cells can divide into large mitotically active pre-B cells and small non-dividing pre-B cells
• Both large and small pre-B cells express Igμ heavy chains in their cytoplasm (cμ = ½ mIgM) and the pre-B cell receptor complex on their surface (mIgM)
• Large pre-B cells have successfully rearranged their Ig heavy chain genes
• As these cells pass from large to small pre-B cell group they begin to rearrange their Ig light chain genes and upregulate RAG-1 and RAG-2
• Pre-B cells also express IL-7R, which when bound by IL-7, stimulates division and differentiation of the pre-B cell
Downstream IL7/IL7R pathway activates different transcription factors:
• E2A and EBF – activate early B cell genes
• Pax5 – ensures development to B cell lineages by restricting transcription of lineage inappropriate genes
• Sox4 and LEF1 – promote survival and proliferation of pro-B cells
• IRF4 and IRF8 – terminate pre-BCR signaling in IRF4 and promote differentiation to small pre B cells
• Bcl-6 – required for germinal B cell differentiation and generation of memory B cells
• Expression of Blimp-1 suppresses Bcl-6 expression and is required for development of Ig secreting cells and maintenance of long lived plasma cells
o Final stage of B-cell development in the bone marrow is the immature B cell stage
• Immature B cells have successfully rearranged their light chain genes & express IgM
• Downregulation of RAG-1 and RAG-2 has occurred and as they develop further, they begin to express both IgM and IgD on their surface
• These naïve mature B cells are then able to exit the bone marrow and migrate into the periphery once they express BCR (IgM and/or IgD)
o Once these naïve mature B cells exit the bone marrow, they go through a transition phase where they undergo negative and positive selection
• Negative selection of many self-reactive B cells occurs in the bone marrow (clonal deletion) and limits the development of antibody-mediated autoimmunity
If immature B cells that express mIgM recognize self-antigen they undergo either apoptosis or editing of their light chain genes that (when combined with heavy chain) does not recognize self-antigen
However, negative selection of some self-reactive B cells occurs in the periphery due to not all self-antigen being present in the bone marrow (MOA under investigation)
o Peripheral Populations of B Cells:
• Follicular (B-2) B cells → ”normal B cells” – bind antigen; differentiate into plasma and memory cells
• B-1 B cells → only express IgM on surface; don’t receive T cell help although they respond to T independent antigen; produce high levels of IgM
• Marginal zone B Cells (spleen) → could be self-renewing; not sure if they go through somatic hypermutation; primarily express IgM, some IgG
o Cytokines Required for B Cell Development:
• IL-7 promotes B cell lineage development
• Blys (B-lymphocyte stimulator) signals through BR3 and is important for survival B cell from pre B cell stage and onwards
• IL-4, IL-3 and low-molecular weight B cell growth factor (L-BCGF) important for initiating process of B cell differentiation

Question 8

TdT and B220 expression:

Answer 8

• Early pro B cell: TdT
• Intermediate pro-B cell: TdT and B220 (glycosylated state of CD45R)
• Late pro-B cell: B220, downregulated TdT
Note, B220 remains expressed on the B cell surface throughout the remainder of maturation

Question 9

pro b-cell expression

Answer 9

• As pro-B cells progress through this stage, they rearrange their Ig heavy chain genes and express CD43 (leukosialin, an adhesion factor to bind bone marrow stromal cells), CD19 (BCR co-receptor, works with CD21 and CD81), RAG-1 and RAG-2
• Pro-B cells also express c-Kit which binds to stem-cell factor expressed on bone marrow stromal cells which induces the pro-B cell to proliferate and differentiate into pre-B cells

Question 10

pre-B cell stage

Answer 10

o As late pro-B cells pass into the pre-B cell stage, they downregulate TdT, RAG-1 & 2, and CD43
o Pre-B cells can divide into large mitotically active pre-B cells and small non-dividing pre-B cells
• Both large and small pre-B cells express Igμ heavy chains in their cytoplasm (cμ = ½ mIgM) and the pre-B cell receptor complex on their surface (mIgM)
• Large pre-B cells have successfully rearranged their Ig heavy chain genes
• As these cells pass from large to small pre-B cell group they begin to rearrange their Ig light chain genes and upregulate RAG-1 and RAG-2
• Pre-B cells also express IL-7R, which when bound by IL-7, stimulates division and differentiation of the pre-B cell
Downstream IL7/IL7R pathway activates different transcription factors:
• E2A and EBF – activate early B cell genes
• Pax5 – ensures development to B cell lineages by restricting transcription of lineage inappropriate genes
• Sox4 and LEF1 – promote survival and proliferation of pro-B cells
• IRF4 and IRF8 – terminate pre-BCR signaling in IRF4 and promote differentiation to small pre B cells
• Bcl-6 – required for germinal B cell differentiation and generation of memory B cells
• Expression of Blimp-1 suppresses Bcl-6 expression and is required for development of Ig secreting cells and maintenance of long lived plasma cells

Question 11

immature B cell stage

Answer 11

o Final stage of B-cell development in the bone marrow is the immature B cell stage
• Immature B cells have successfully rearranged their light chain genes & express IgM
• Downregulation of RAG-1 and RAG-2 has occurred and as they develop further, they begin to express both IgM and IgD on their surface
• These naïve mature B cells are then able to exit the bone marrow and migrate into the periphery once they express BCR (IgM and/or IgD)

Question 12

naïve mature B cells

Answer 12

o Once these naïve mature B cells exit the bone marrow, they go through a transition phase where they undergo negative and positive selection
• Negative selection of many self-reactive B cells occurs in the bone marrow (clonal deletion) and limits the development of antibody-mediated autoimmunity
If immature B cells that express mIgM recognize self-antigen they undergo either apoptosis or editing of their light chain genes that (when combined with heavy chain) does not recognize self-antigen
However, negative selection of some self-reactive B cells occurs in the periphery due to not all self-antigen being present in the bone marrow (MOA under investigation)

Question 13

o Peripheral Populations of B Cells

Answer 13

• Follicular (B-2) B cells → ”normal B cells” – bind antigen; differentiate into plasma and memory cells
• B-1 B cells → only express IgM on surface; don’t receive T cell help although they respond to T independent antigen; produce high levels of IgM
• Marginal zone B Cells (spleen) → could be self-renewing; not sure if they go through somatic hypermutation; primarily express IgM, some IgG

Question 14

• Follicular (B-2) B cells

Answer 14

”normal B cells” – bind antigen; differentiate into plasma and memory cells

Question 15

• B-1 B cells

Answer 15

→ only express IgM on surface; don’t receive T cell help although they respond to T independent antigen; produce high levels of IgM

Question 16

• Marginal zone B Cells

Answer 16

(spleen) → could be self-renewing; not sure if they go through somatic hypermutation; primarily express IgM, some IgG

Question 17

o Cytokines Required for B Cell Development:

Answer 17

• IL-7 promotes B cell lineage development
• Blys (B-lymphocyte stimulator) signals through BR3 and is important for survival B cell from pre B cell stage and onwards
• IL-4, IL-3 and low-molecular weight B cell growth factor (L-BCGF) important for initiating process of B cell differentiation

Question 18

• IL-7

Answer 18

promotes B cell lineage development

Question 19

• Blys

Answer 19

(B-lymphocyte stimulator) signals through BR3 and is important for survival B cell from pre B cell stage and onwards

Question 20

• IL-4

Answer 20

important for initiating process of B cell differentiation

Question 21

IL-3

Answer 21

important for initiating process of B cell differentiation

Question 22

L-BCGF

Answer 22

important for initiating process of B cell differentiation

Question 23

o Abnormalities in B Cell Development:

Answer 23

• XLA Immunodeficiency highlights the importance of pre-BCR signaling in B cell development as a mutation in the Btk gene (Bruton’s tyrosine kinase) results in a block at the pro-B cell to large pre-B cell transition
Defect in Btk is evidenced through very few circulating B-cells and negligible serum immunoglobulin (Ig)
XLA accounts for 85% of agammaglobulinemia cases
• Other causes characterized by mutations in: μHC, λ5, Igα (CD79a), Igβ (CD79b), and BLNK (SLP-65), all affecting pre-BCR functions
• Common variable Immunodeficiency (CVID) impacts B cell development in later stages
CVID manifests in reduced serum Ig, memory B cells, class switch recombination, and B cell activation
Mutations in CD40L on T cells, B cell surface receptor CD19, activated T cell co-stimulatory molecule ICOS, and TACI (another receptor for Blys) have been implicated and identified in CVID

Question 24

• XLA Immunodeficiency

Answer 24

highlights the importance of pre-BCR signaling in B cell development as a mutation in the Btk gene (Bruton’s tyrosine kinase) results in a block at the pro-B cell to large pre-B cell transition
Defect in Btk is evidenced through very few circulating B-cells and negligible serum immunoglobulin (Ig)
XLA accounts for 85% of agammaglobulinemia cases
• Other causes characterized by mutations in: μHC, λ5, Igα (CD79a), Igβ (CD79b), and BLNK (SLP-65), all affecting pre-BCR functions

Question 25

• Common variable Immunodeficiency

Answer 25

CVID) impacts B cell development in later stages
CVID manifests in reduced serum Ig, memory B cells, class switch recombination, and B cell activation
Mutations in CD40L on T cells, B cell surface receptor CD19, activated T cell co-stimulatory molecule ICOS, and TACI (another receptor for Blys) have been implicated and identified in CVID

Question 26

• T-independent (TI) antigen o B Cell Activation

Answer 26

small number of antigens can activate B cells without MHC II-restricted T cell help; these TI antigens activate B1 B cells (express CD5)
Two groups of TI antigens:
• TI-1 = bacterial cell wall components – LPS (gram-neg bacteria)
• B1 B cell binds to LPS either through TLR4 or BCR
• TLR4 = nonspecific (polyclonal activation) – found on multiple different cells (immature and mature B cells)
• BCR = specific (clonal activation)
• Only IgM is produced in response to this stimulation
• TI-2 = large polysaccharides – Ficoll, dextran, flagellin, poliomyelitis virus
• Many TI antigens are PAMPs that can be recognized by TLRs
• B1 B cells bind to TI-2 antigens through cross-linking of BCR which leads to clonal activation
• TI-2 antigen only stimulates mature B cells through use of BCR
• Mostly IgM is produced in response to this stimulation
• CD4+ Th2 cells can be involved to produce cytokines for a full B cell response that can include class-switching
• Overall, TI antigens produce poor memory but they are able to replenish themselves

Question 27

oT-dependent (TD) antigen B Cell Activation

Answer 27

most antigens depend on both T and B cells recognizing antigen in a linked fashion
B cells also bind Th2 cells directly through MHC Class II molecules and exogenous proteins
• Note, Th1 cells interact with B cells via cytokines and promote B cell division and differentiation, thus they indirectly interact with B cells
Antigen binding to either 1) 2 BCRs or 2) a BCR + co-receptor (MHC Class II – antigen complex) is required for intracellular signaling:
• 1) If cross-linked, then CD79a/b activates signaling through their ITAM repeats and a cascade of signaling leads to eventual activation of NFkB and G proteins that activate transcription factors Rho, Rac, and Ras
• 2) Instead of cross-linking, the BCR has a co-receptor which requires complement (C3b) to bind CD21/19/81 (BCR co-receptor complex) and allows for mIgM and secreted IgM to be made; however, class switching cannot occur
Upon Th2 cell binding to a B cell, the Th2 cell produces IL-4, 5, 6, 10, 13 to cause the B cell to divide and differentiate into a plasma cell
Different B and T cell surface molecule interactions result in different outcomes:
• Antigen-receptor transduction events (MHC II – antigen; BCR – antigen): tyrosine phosphorylation and phosphoinositide breakdown; regulate LFA-1 affinity for ICAM-1 and 3
• CD28 (CTLA-4) when bound to B7 (CD80/86) causes a unique signal within the T cell
• In later stages of this response, CLTA-4 can supplant CD28 to cause its downregulation
• Stimulation of CD40 in the B cell is the most potent activating signal
• Exchange of soluble interleukins occurs and binding results on corresponding receptors on the other cell
o Ultimately, TD antigen will result in generation of memory B cells which are responsible for the primary and secondary antibody responses found during/after infections
• Once the B and Th2 cell bind or B cells interact with free antigen, the B cell complex will migrate to the germinal center of a lymph node
If B cells do not bind to free antigen or receive help from T cells, SHM dies by Fas-independent apoptosis (only live for 3 weeks unless they bind antigen)
However, if the B cell binds to Th2 cell it makes Bcl-2 which protects the cell from apoptosis
• The activated B cell will undergo somatic hypermutation which leads to either better or worse antigen binding abilities for the B cell (worse binding is selected for apoptosis)
• Following somatic hypermutation. the B cell can bind to a T cell through CD40/CD40L to become a plasma or memory B cell
o Summary:
• B cell development occurs in the bone marrow and does not require contact with antigen
• During this time, B cells rearrange the genes for their heavy and light chains as well as synthesize surface IgM which acts as their initial BCR
Initial BCR includes: membrane-bound IgM (mIgM) and signaling chains Igα and Igβ (CD79a and b) used in intracellular signaling
• Immature transitional B cells exit the bone marrow and enter the periphery where they undergo further maturation in secondary lymphoid organs (spleen, etc.)
If these cells do not encounter antigen, they will soon die within a few weeks via apoptosis (do not express Bcl-2 protein)
If these cells do encounter antigen, they undergo activation, proliferation, and differentiation → plasma and memory B cells

Question 28

• Describe the process of T cell maturation in the thymus.

Answer 28

o T cells, once they leave the bone marrow, they require further education in the thymus
o Maturing T cells are called thymocytes and they undergo positive and negative selection
• Thymic stromal cells express Notch which is required for thymocyte development (much like c-Kit is involved in B cell development in bone marrow)
• No Notch = no T cell development
• Takes approximately 3 weeks to pass through the thymus
o Early thymocyte development results in T cells being CD4 and CD8 negative (double negative, DN) and require CD44 for relocation to the thymus, c-Kit for replication, and CD25 (IL-2R) for IL-2 driven replication
o Progressing through the different DN stages is evidenced by changes in the TCR: α and β chains are selected
o The T cell progresses through the DN stages to become a double positive (DP) cell which expresses both CD4 and CD8
• It is within the DP stage that the T cell undergoes positive and negative selection
Positive selection = self-restriction (recognize self-MHC molecules)
Negative selection = self-tolerant (cannot respond to self-antigens)
• Only 2-5% of T cells make it through this selection process
• It is unclear how the T cell goes from being DP to expressing only CD4 or CD8 (single positive, SP) – multiple theories include:
Instructive model: bind to APC and are told what T cell to become
Stochastic model: randomly decrease expression of one of the surface markers
Kinetic signaling model: differences in IL-7 signaling

Question 29

• Describe the process of T cell activation and know which cells and molecular signals are involved in this process.

Answer 29

o Binding events to activate T cells:
• Binding of one TCR is all that is required for T cell activation.
• Maximum activation ~10 TCRs binding at once.
• Two binding interactions (TCR + co-stimulatory interaction, i.e. CD28/B7) plus cytokines required for full activation of T cells
o T cells have a high affinity IL-2R which when bound by IL-2 results in T cell enlargement and several rounds of division to create mostly effector T cells and some memory T cells
• Usually 2-3 divisions occur per day for 4-5 days → making 256 – 32,768 T cells
o After antigen binding to APCs (DCs can have cross-presentation) many genes are activated:
• Immediate genes = within 30 minutes → transcription factors c-Fos, c-Myc, c-Jun, NFAT, and NF-kB.
• Early genes = within 1 to 2 hours → Encode IL-2, IL2-R, IL-3, IL-6, IFNγ, others.
• Late genes = more than 2 days later → adhesion molecules.
o If a T cell comes across an APC and the TCR binds to the right antigen and co-receptor (MHC – CD), but CD28 cannot bind B7 then this triggers an anergic reaction which causes the T cell to become desensitized and can no longer be activated (T cell dies within a few days)
o Superantigens can bind both MHC and TCR outside of the antigen groove of the complexes and initiates a non-specific interaction to stimulate many T cells of different antigenic specifications
• Activation of many T cells results in induction of IFNγ and TNFα → toxic shock
• Superantigens appear to work without the need of B7 since they bind to CD28 to provide the co-stimulatory signal

Question 30

anergic reaction

Answer 30

o If a T cell comes across an APC and the TCR binds to the right antigen and co-receptor (MHC – CD), but CD28 cannot bind B7 then this triggers an anergic reaction which causes the T cell to become desensitized and can no longer be activated (T cell dies within a few days)

Question 31

Superantigens

Answer 31

can bind both MHC and TCR outside of the antigen groove of the complexes and initiates a non-specific interaction to stimulate many T cells of different antigenic specifications
• Activation of many T cells results in induction of IFNγ and TNFα → toxic shock
• Superantigens appear to work without the need of B7 since they bind to CD28 to provide the co-stimulatory signal

Question 32

• Describe the process of T cell differentiation and review the various subsets of T cells that are produced; what their functions are in immunity; what cytokines they produce; and what cytokines they respond to.

Answer 32

o Naïve CD4 T cells are induced to form different forms of T helper cells via use of different polarizing cytokines which activate different transcription factors resulting in different cytokine products
• The cytokine products determine the function of the T helper effector cells
o Antigenic history can also dictate (to a degree) the path of T cell differentiation:
• Cell-mediated immunity: most viruses, some bacteria, fungi
• Humoral immunity: extracellular parasites (worms)

Question 33

• Describe the role of cell mediated immunity within an immune response.

Answer 33

o Cell mediated immunity recognizes pathogen-infected cells or cells that have undergone genetic alterations (tumor cells) and kills them
o Mediators of cell-mediated immunity:
• Antigen non-specific effector cells: NK cells, macrophages, neutrophils, eosinophils
• Antigen specific effector cells: CD8+ T cells (CTLs) and CD4+ T cells

Question 34

• Describe the mechanism of activation used by cytotoxic CD8+ T cells.

Answer 34

o Naïve CD8+ T cells are called CTL precursors (CTL-P) in their immature state
o CTL-P matures after being activated by an interaction with Th1 or 17 CD4+ T cell
o Maturation requires three sequential signals:
• 1. Antigen-specific signal = TCR recognizes proper peptide
• 2. Co-stimulatory signal = example CD28:B7
• 3. IL-2 secretion from Th1 CD4+ T cell = results in proliferation and differentiation; thus CTL-P can become a fully active CTL

Question 35

o Generation of Effector CTLs:

Answer 35

• Sequential: Th1 or Th17 CD4+ T cell interacts with a licensed APC (CD40L of T cell binds to CD40 on APC or through a pathogen PAMP presented on APC to a TCR on the T cell)
Presentation of antigen to a CTL-P through MHC I can only occur once the APC has “found” pathogen or has been told it has “found” antigen through T Cell → indicates the APC is licensed
Infection of DCs by virus can also assist in antigen presentation associated with MHC I
• Simultaneous: Th1 remains bound to the APC and the CTL binds as well resulting in cross-presentation

Question 36

CD8+ T cell Maturation

Answer 36

• Naïve CTL-P:
Does NOT express IL-2 or IL-2R (express upon activation via antigen presentation)
Express CD45RA (isoform of CD45) and low levels of CD2 and LFA-1 (adhesion molecules)
Do NOT have cytotoxic activity (no granules)
• Mature CTL:
Express high affinity IL-2R and requires high levels of IL-2 to proliferate, CD45RO (isoform of CD45), high levels of CD2 and LFA-1
Exhibit cytotoxic activity (have granules – granzymes and perforins)
• Memory CTLs:
May not require Th1 CD4+ T cell help to reactivate
Requires only low levels of IL-2 (produced by activated CTLs) for memory CTLs to become mature effector CTLs

Question 37

o CTL Killing:

Answer 37

• The TCR-CD3 complex on CTL recognizes peptide-MHC I complex on target cell
• LFA-1 on CTL binds to ICAMs on target cell
• Antigen activation converts LFA-1 from low affinity to high affinity state for better binding (after ~5-10 minutes, LFA-1 returns to low affinity state allowing dissociation of CTL from the target cell)
• Mechanisms of CTL killing:
1. Perforin and Granzyme Secretions:
• Perforins form a pore on target cell membranes. Monomers of perforin for pores similar to C9 from complement making MAC
• Granzymes activate apoptosis by cleaving caspases in both intrinsic and extrinsic pathways
2. Fas ligand protein on CTL surface interacts with Fas Death Receptor (“kiss of death”)
3. CTLs kill target cells through TNFα production and secretion

Question 38

• Describe mechanism of activation and method of killing used by NK cells.

Answer 38

o NK cells constitute 5-10% of population of circulating lymphocytes
o Major role of NK cells: killing virus-infected cells, intracellular pathogen-infected cells, and tumor cells
o Induced by: IFN-α, IFN-β, IFN-γ, TNF-α, and IL-15
o Produce cytokines such as IFNγ
• IFNγ – activate M1 macrophages and NK cells; tilt immune response toward Th1 cells by inhibiting Th2 cells and inducing IL-12 production by macrophages & DCs
o NK cells are an early defense against viruses
o Comparison of NK cells and CTLs:
• NK cells express CD16 and FcγRIII
• NK cells are NOT educated in the thymus, nor do they undergo rearrangement of receptor genes (don’t make TCR or CD3)
• NK cell killing is similar to CTL killing; however, it is not MHC restricted
1. Perforin and granzyme release from granules
2. FasL expressed on surface can kill cells expressing Fas
3. TNFR expressed on surface of target and NK cells secrete TNFα

Question 39

o NK Cell Receptors:

Answer 39

• Two categories that are inhibitor or non-inhibitory (activating):
Lectin-like Receptors: binds proteins
• Inhibitory – receptor has CD94 and NKG2A binding segments; also have intracellular ITIM segment
➢ CD94-NKG2A (NK receptor) binds to HLA-E ligand → inhibitory
• Non-inhibitory – CD94 and NKG2C binding segments; also have intracellular Lysine (K) residue
➢ Inhibition receptors have a higher affinity for their ligand than non-inhibitory receptors; takes more work to activate an NK cell
Immunoglobulin-like receptors (KIR = killer cell Ig-like receptors): binds to most MHC class I molecules
• Inhibitory – receptor has KIR-2D long and KIR-3D long binding segments with intracellular ITIMs
• Non-inhibitory – receptor has KIR-2D short and KIR-3D short binding segments with two intracellular Lysine (K) residues
Some cells have activating and inhibitory signals that occur simultaneously:
• ITIMs interact with SHP-1 and ZAP70 to block Ca2+ signaling and cytoskeletal rearrangement
• Inhibitory signals tend to override activating signals

Question 40

Lectin-like Receptors

Answer 40

binds proteins
• Inhibitory – receptor has CD94 and NKG2A binding segments; also have intracellular ITIM segment
➢ CD94-NKG2A (NK receptor) binds to HLA-E ligand → inhibitory
• Non-inhibitory – CD94 and NKG2C binding segments; also have intracellular Lysine (K) residue
➢ Inhibition receptors have a higher affinity for their ligand than non-inhibitory receptors; takes more work to activate an NK cell

Question 41

Immunoglobulin-like receptors

Answer 41

(KIR = killer cell Ig-like receptors): binds to most MHC class I molecules
•	Inhibitory – receptor has KIR-2D long and KIR-3D long binding segments with intracellular ITIMs
•	Non-inhibitory – receptor has KIR-2D short and KIR-3D short binding segments with two intracellular Lysine (K) residues

Question 42

• Opposing signals of NK Cell Killing

Answer 42

If antigen is recognized by the NK cell and the target cell expresses:
• High levels of MHC class I → target cell is NOT killed
• Low levels of MHC class I → target cell is killed
➢ When the inhibitory receptor does not interact with MHC I, it will allow the positive ligand-activating receptor signal to kill the target cell
➢ When this killing occurs, it is extremely similar to CTL killing:
• 1) Perforins and granzymes
• 2) Fas death receptor activation
HLA-E presents MHC leader peptide to NK cells which can act in place of MHC I presentation to allow the cell to NOT be killed
• Leader peptides from MHC class I molecules (yellow bar) are loaded onto HLA-E molecules in the endoplasmic reticulum, a process that requires TAP transporters and tapasin to assemble functional HLA-E molecules.
• These are presented at the cell surface for review by CD94 receptors on NK cells
• The MHC class I molecules meanwhile present antigenic peptides from cytoplasmic proteins that have been transported into the endoplasmic reticulum.
• These complexes are presented to the TCR on CD8+ CTLs
• This mechanism serves as a protective measure for normal cells from NK cells when normal cells have a difficulty getting MHC I out on their surface
➢ Could this be a mechanism of viral infection too???

Question 43

• NK Cells and CTLs complement each other’s function

Answer 43

NK Cells
kill cells that mask the presence of foreign antigen on MHC class I
Express Lectin-like receptors and KIR; variant of TCR that recognizes glycolipids
No memory cells

CTLs
kill cells that express foreign antigen on MHC I
Express TCR and CD3
Memory cells

Question 44

o NK Cell Development

Answer 44

• Surface markers that are required for the NK cells effector functions come in a later stage of development
CD161, NKp46, CD56, CD94, CD122, KIR; CD38 and CD117 (aka c-Kit) decline

Question 45

• Describe the method of killing associated with antibody-mediated cellular cytotoxicity.

Answer 45

o Effector cells associated with ADCC: NK cells, macrophages, monocytes, neutrophils, eosinophils
o Effector cells bind antigen via antibody, through the Fc receptor
o Killing is mediated by cytolytic enzyme release by macrophages, neutrophils, and eosinophils
• TNFα released by NK cells, monocytes, and macrophages.
• Perforin release by NK cells and eosinophils
• Granzyme release by NK cells

Question 46

• Understand the main features of chronic leukemias (as opposed to acute leukemias).

Answer 46

o Malignant proliferation of myeloid cells (not blasts, but maturing cells) in blood, bone marrow
o Four disorders: CML, PV, ET, MF
o Occur only in adults
o Long course

Question 47

o Chronic myeloid leukemia

Answer 47

neutrophils proliferating
•	Stages of CML:
•	Chronic Phase:
o	Stable counts 
o	Easily controlled
o	3-4 years (untreated)
•	Accelerated Phase (50% of chronic phase CMLs progress to the accelerated phase):
o	Unstable counts
o	Blast crisis within 6-12 months
•	Blast crisis (50% of chronic phase CMLs progress to blast crisis):
o	Acute leukemia (blast count is > 20%) – although usually get ALL (strange!!) – due to mutation in a stem cell which at the end stages becomes lymphoid
o	High mortality

Question 48

o Polycythemia vera

Answer 48

red cells proliferating (“Polycythemia” = ↑ red cell mass)
• Two Different Types:
• Primary (intrinsic myeloid cell problem)
• Secondary (due to increased erythropoietin)
• Diagnostic Criteria:
• A criteria (major):
o *A1 ↑ red cell mass
o *A2 Normal O2 saturation
o A3 Splenomegaly
• B criteria (minor):
o B1 Thrombocytosis
o B2 ↑ WBC without infection
o B3 ↑ LAP – not sure why…
o B4 ↑ B12 – abnormal B12 metabolism
• Genetics: JAK-2 in PV
• Normal JAK-STAT pathway → cell signaling important in many different cell types
• JAK-STAT in PV → activity increased in PV and cells grow on their own

Question 49

o Essential thrombocythemia

Answer 49

platelets proliferating

Question 50

o Myelofibrosis

Answer 50

everything proliferates!

Question 51

o Common Features to All Four CML Disorders:

Answer 51

• Occur only in adults
• Long clinical course
• ↑ WBC with left shift
• Hypercellular marrow
• Big spleen
• May evolve into acute leukemia
• Mutated tyrosine kinases

Question 52

• Genetic changes underlying the development and progression of CML

Answer 52

o Initiating event known at molecular level: chromosomal translocation t(9;22)
• Chromosome 22 = IgL chain (lambda); chromosome 9 = ABL gene (tyrosine kinase)
o Oncogenic mechanism known: genomic translocation creates new gene encoding constitutively active tyrosine kinase
o Targeted molecular therapy based on mechanism: tyrosine kinase inhibitors, Imatinib

o Outcome of normal neutrophil differentiation:
• Self-renewal and proliferation are tightly regulated by extracellular signaling from bone marrow
• Appropriate number of mature neutrophils are produced

Question 53

o Changes in normal neutrophil differentiation during CML Chronic Phase

Answer 53

• BCR-ABL1 genomic translocation in hematopoietic stem cell
• Creates genetic chimera of parts of BCR (breakpoint cluster region) gene and ABL1 (ableson tyrosine kinase) gene
• BCR-ABL1 fusion protein is a constitutively active tyrosine kinase that activates proliferation and blocks apoptosis in absence of extracellular signals
o BCR-ABL1 progenitor cells proliferate more, survive longer
o BCR-ABL1 progenitor cells have the opportunity to acquire more mutations which can make cells more oncogenic
o However, constitutively active BCR-ABL1 DOES NOT confer the ability to self-renew
• BCR-ABL1 progenitor cells DO continue to differentiate though
• Double stranded breaks occur in two chromosomes at specific breakpoints
o Two most common:
• p210 found in CML, most common
• p190 found in some ALL, less common
o Mechanism used is non-homologous end-joining (NHEJ) – ends of different chromosomes are jammed together (not able to repair the damage)
• Normal functions of the different genes:
o BCR – role in inhibition of some inflammatory responses
• Functional domain:
• normal – Ser/Thr kinase
• oncogenic – coiled-coil domain, tyrosine 177
o ABL – role in DNA repair, cytoskeletal organization
• Function domain: tyrosine kinase
• Kinase is held inactive unless activated by external signals (growth factors)
• Myristate attachment to ABL1 maintains inhibition
o Neither BCR or ABL are oncogenic alone; both ABL1 and BCR-ABL1 are tyrosine kinases (which are regulated by intracellular signaling pathways)
• Important differences:
• 1. ABL1 (normal cells) is inactivated unless activated by extracellular signaling
• 2. ABL1 and BCR-ABL1 activate different intracellular signaling pathways
• Differences due to structural changes:
• Coiled-coil domain is added from BCR promotes dimerization, dimerization is necessary for activation
• Myristate attachment site lost from ABL1 myristate necessary for autoinhibition of ABL1 tyrosine kinase activity
• Tyrosine-177 (Y-177) is added from BCR phosphorylation of Y-177 creates a new binding site for intracellular signaling proteins
• Oncogenic signaling: BCR-ABL1 does NOT require cell signaling to initiate its function, whereas ABL1 requires cell signaling to initiate its normal function
o BCR-ABL1 phosphorylation of Y177 serves as a binding site for adaptor proteins which is a starting point for several oncogenic signaling pathways
o In presence of BCR-ABL, JAK-STAT is activated independently of JAK2 itself → allows Stat5 to translocate to nucleus to allow transcription of anti-apoptotic genes
• Reason why progenitor cells proliferate independently of external signals
o Different pathways activated from BCR-ABL1:
• CRK-P → PI3 kinase → proliferation, anti-apoptosis, cell growth (normal ABL1 function)
• GRB-P →1) Ras–MAPK, 2) JAK/STAT → transcription for proliferation and survival
• Outcome:
• Expansion of progenitor and committed cells → increased proliferation and survival of progenitor cells
• But mature cells are still produced
• Disease is relatively mild
• Mutations arise in hematopoietic stem cell but is passed down to all progeny
• Have the opportunity to acquire more mutations

Question 54

o Changes in normal neutrophil differentiation during Accelerated/Blast Phases of CML

Answer 54

• Blast phase: GMP acquires ability to self-renew and acquires blocks to differentiation → huge expansion of blasts (30% extramedullary)
• BCR-ABL1 alone does not cause progression from chronic to blast phase:
• Additional mutations are needed
• Granulocyte Myeloid Progenitor (GMP) acquires self-renewal capability
o Mutations activate self-renewal signaling pathways such as beta catenin
• GMP lose ability to differentiate due to mutations
• Mechanism by which imatinib blocks CML progression.
o Imatinib specifically inactivates ABL1 kinase by blocking ATP binding
• BCR-ABL mutant cells depend on BCR-ABL for proliferation and anti-apoptotic signaling and inactivation of BCR-ABL results in apoptosis of the mutant cells
• Loss of BCR-ABL allows normal cells to repopulate
o Imatinib is extremely effective!
• 5 year survival rates for newly diagnosed chronic phase CML is 85-90%
• Natural history without treatment, chronic phase lasts for 3-4 years
o Limitations of BCR-ABL1 inhibitors:
• 1. Secondary resistance due to changes in BCR-ABL protein
• 2. BCR-ABL1 inhibitors are not effective against blast phase disease due to additional mutations to progress to this stage of the disease
• 3. CML stem cells are innately resistant to tyrosine kinase inhibitors
• Imatinib must be taken for life → increased resistance susceptibility

Question 55

• Describe the mechanisms of action and side effects of the currently approved signal transduction inhibitors, and explain the success of imatinib vs. gefitinib.

Answer 55

page 21

Question 56

• Bcr-abl:

Answer 56

non-receptor tyrosine kinase disregulated by the translocation of its gene from chromosome 9 to chromosome 22 in most patients with CML

Question 57

• c-kit

Answer 57

tyrosine kinase altered in gastrointestinal stromal tumors (GIST)
• Platelet-derived growth factor (PDGF) receptor (weak)

Question 58

• DASATANIB

Answer 58

also targets Src, a tyrosine kinase whose expression is upregulated in several types of cancer
• May be anti-metastatic in epithelial-derived tumors

Question 59

• Describe the major differences between Hodgkin and non-Hodgkin lymphoma.

Answer 59

o	Hodgkin Features:
•	Younger patients, good prognosis
•	Contiguous spread
•	Five subtypes; stages are more important
•	Reed-Sternberg cell
o	Non-Hodgkin Features:
•	Malignant proliferation of lymphoid cells (blasts or mature cells) in lymph nodes
•	Skips around in body (not predictable)
•	Many subtypes
•	Most are B cell

Question 60

• Describe the features of a benign lymph node enlargement (as opposed to a malignancy in a lymph node). Which is the most common?

Answer 60

o Causes of Lymphadenopathy:
• Most common cause overall: benign reaction to infection
• Most common malignant cause: metastatic carcinoma