Notes Test 3

Notes Test #3

***March 25 Paper

I. Interferons

*Cytokines
*Both type I and II detect viruses -> transcription and translation of other cytokines, inflammatory mediators (IL-12 and IL-6)

a. Type 1 IFNs: IFN alpha and beta
*Recognize -> response to viral parts inside the cell
*Viral motifs in cell cytoplasm (if cell is infected)
*In vacuoles (in uninfected dendritic cells) - interact with TLR 3,6,7,8 and 9
*Released in response to viruses

b. Type 2 IFNs: IFN gamma
*Similar in action to type I IFNs
*but also recognizes other intracellular pathogens besides viruses

IFN recognition triggers JaK-stat pathway: phosporilation cascade.
 Release cytokines like IL-1, TNF, IL-6 and IL-12

I. Effects of Interferons

*Expression of inflammatory cytokines
*Expression of anti-viral enzymes

a. Stop protein production (Antiviral enzymes)
*Transcription: mRNA (RNase: destroys RNA, RNase L. degrades mRNA and rRNA)
*Translation:amino acid chain (Protein that disables cell’s own initiation factor -> stopping initiation of transcription) (produces mRNA which is destroy by RNase) the initiation factor is PKR which needs double stranded RNA to work

b. Stopping viral budding from host cell
*Mx proteins stop blebs of cell membranes from pinching off

c. Viral evasion of IFNs effects
*Flu virus covers double stranded RNA with a protein
*Poxviruses- stop phosphorylation cascade that -> antiviral gene expression

d. IFNs as disease treatments
*IFN-alpha used to treat Hepatitis B and C
*Virally- caused cancers: ex: Kaposi’s (Sarcoma caused by HIV)

II. Induction of apoptosis

a. p53 pathway
*Phosphorilation cascade
*Results in mitochondrial release of ions and proteins
*Cell death thru apoptosis
*Triggered by DNA replication out of sync with mitosis

b. Fas ligand and TNF-receptor binding
*Fas binds to Fas ligand and also apoptosis
*TNF receptor- TNF binds to TNF-receptor if cell has stopped protein production, apoptosis results

c. Viral evasion of apoptosis
1. Human Papilloma Virus (HPV) types 16 and 18
*The types 16 and 18 make E6, a protein that binds to p53.
*E6-p53 complex bound by ubiquitin. Ubiquitin binding to any protein marks it for destruction.
*Bcl2-> negative feedback
-turns off apoptosis pathway
-HPV 16 and 18 make transcription factor that increase expression of Bc12

III. Disabling viral nucleic acids

a. Cytosine deaminases
*Enzyme that removes NH from cytosine Cytosine binds Uracil
-this causes a DNA mutation
-virally infected cells produce cytosine deaminases which are package into capsid retroviruses
*HIV makes vif which ubiquitination and degradation of cytosine and deaminases
b. RNAi<- interference
*In response to double stranded RNA, cell makes enzyme (“Dicer”) which cleaves dsRNA into tiny fragments
*DS RNA fragments recognized by set of other proteins which then destroy any ss RNA homologous to ds RNA fragments.

**April 1

I. A review of the role of T-cells in adaptive immunity
*Lympho tissue ex. Spleen and lymph

a. Adaptive vs Innate immunity
*Allow our cells to recognize chemicals
*Allows immune system to recognize microbial chemicals that aren’t shared by many kinds of microbes
*Takes more time
*Innate prolly more important than adaptive is always on and not flexible

b. T-cell receptors
*Constant region- same as amino acid sequence in all T-cells.
*Constant region- attaches to cell membrane, helps with signaling transduction from outside to inside of cell
*Variable region attaches to antigen->different amino acid sequence in each T-cell

c. CD8+ T-cells
ii. Host vs graft disease
*Lots of different MHC-I’s among folks
*Foreign MHC-I is recognized by CD8 T-cell receptor as MHC-1 presenting foreign antigen
*So CD8+, cytotoxic T-cell kills “virally infected cell”

II. Dendritic cells <- last few days or few weeks

a. Immature
*Hanging out in tissues
*Globular shape
*Immature recognizes TNF
*Constantly sampling environment thru phagocytosis and lots of macropinocytosis

b. Mature
*Spontaneously matures become star-shaped, expresses CCR7-> migration to lymph nodes
*In lymph nodes, mature dendritic cell presents antigens from cell debris to T-cells
*Thought that presentation of antigens by mature, but not “activated” dendritic cells helps maintain tolerance to own antigens

c. Mature, activated
*Immature dendritic cells sampling environment
*Detects microbial components via TLR’s or pro-inflammatory cytokines (IL1 and TNF) via receptors for them (leads to: leads to upgrade in phagocytosis and macropinocytosis)
MHC II and some MHC 1
Express gene
Expression of costimulatory molecule (molecule that help activate T-cells in here B7 family is the costimulatory family)
B7 costimulation of T-cells is how T-cells “know” this dendritic cell is presenting foreign and not self antigens.

III. Major histocompatibility complex (MHC) molecules

a. Classical vs non classical MHC’s
*Directly involve in presenting antigen to T-cell receptors (MHC I and MHC II)
*Non-classical don’t present antigens but rather their cytoplasmic protein that help load antigens onto classical MHC’s

b. Diversity of MHC’s
*Lots of alpha and beta chain alleles: MHC components very polymorphic
*> 500 alpha chain alleles, so no one likely to be homozygous for alpha chain alleles
ii. Evolution of MHC diversity:
*Advantageous to have lots of variability in MHC antigen-binding sites because this allows a population of humans to bind more variable antigens, fight wider away of pathogens

c. Structure of MHC I
i. Alpha chains
*Combine to make MHC I
*Alpha 3 attaches MHC I to cell membrane
*Beta 2 microbulin stabilizes alpha chains
ii. Peptide binding groove
*Where peptide (antigen to be presented to T-cell is attached to MHC-1)
*MHC I peptide binding groove binds only 8-10 amino acids in length
iii. Consensus binding

  • A few amino acids in just a few spots seem to be necessary for MHC I peptide binding

d. Structure of MHC II
i. Alpha and Beta chains
*2 alpha chains and 2 beta chains- an alpha and beta interact with cell membrane to form peptide bonding groove
ii. Peptide binding groove
*No consensus anchor sites on peptides
*Can bond peptides (around 20 amino acids in length)

IV. Loading of peptides onto MHC’s

a. “Chaperone proteins”
*Proteins that hold nascent (proteins in correct shape so they will fold correctly)

b. MHC I
*Presents intracellular pathways parts in CD 8+ T-cells
*mRNA translated by ribosomes attached to endoplasmic reticulum
-Alpha chains of MHC I unstable with out peptide bound to peptide binding groove

*So chaperone proteins bind to protect new MHC I until peptide (in cytoplasm of cell) binds to it (because that is where pathogen was, then pathogen proteins cleaved into peptides that fit MHC I)
*Enzymes that cut up pathogen proteins=proteasome<- in cytoplasm
*Proteasome interacts with ER allows transportation of peptides across ER membrane, where can be loaded onto MHC I
*MHC I and peptide blebs off of ER in vacuole, transported to cell surface where vacuole intregates with cell membrane

c. MHC II
*Peptides from extracellular pathogens
*Vacuole containing pathogen peptides fuses with vacuole from lumen of ER containing MHC II which is stabilize by binding CLIP in peptide binding groove
*CLIP MHC II binding weak easily displaced by peptide complementary to peptide binding groove.

V. Antigen presentation by dendritic cells

*TLR or cytokine receptor binding to ligand-> lots of phagocytosis by dendritic cells
*This -> MHC expression
*After phagocytic burst, few rounds of MHC expression, MHC expression shut down
*Then actuvated mature dendritic cells migrates to secondary lymphoid tissues

**April 8

I. T-cell review

a. Maturation in thymus
*Folks who lack thymus (di George Syndrome)
-Can’t make antibodies against intracellular pathogens and hard time making antibodies against extracellular pathogens.

b. Antigen binding
*Presented antigens on MHC’s mainly in secondary lymphoid tissue

c. Co-stimulation with B-7 family molecules
*B-7 family proteins expressed on surfaces of activated mature dendritic cells
*B-7 is a co activator of T-cells helps bind MHC, antigen and T-cell receptor
*B-7 stimulates T-cell to differentiate

d. Migration
*Activated, mature T-cells migrate to B-cell areas of secondary lymphoid tissue where they present antigen (on T-cell receptor) to B-cells, making B-cells
-> Then almost all cloned differentiate T-cells die, a few remain as T-memory cells.

II. Subcategories of CD4+ T-cells
*Differentiation into categories a result of different cytokine concentrations

a. TH 1
*Recruit phagocytes to deep tissues and response to inflammatory cytokines

b. TH 2
*Recruit phagocytes, eosinophils, basophils, and mast cells to epithelia

c. Follicular T-helper (focus the most)
*Interact with B-cells, causing B-cells to become plasma cells, secrete antibody
*This happens in B-cells containing follicles of secondary lymphoid tissue

d. Memory T-cells (hang out in G 6 for decades)
*Left overs after T-cell has
-Recognized an antigen presented in MHC II
-Cloned itself a bunch
-Stimulated B-cells to produce antibodies specific to its antigen
-And after the infection was over most of the clones had died off

III. T-cell receptor structure

a. Alpha and beta chains
*Actively involved in binding to antigen presented on MHC
*Not involved in signal transduction
*Antigen recognition via dimer of alpha and beta chains

b. Signaling chains
*Involved in signal transduction in activated T-cell
*Not variable

IV. T-cell variability

a. Constant region
*As name implies doesn’t vary
*Part of TCR and alpha and beta chains that is attached to cell membrane

b. Variable region
*Composed of alpha and beta chain dimmers
*alpha/alpha, beta/beta, alpha/beta
*Hypervariable region: Where TCR directly interacts with antigen

i. Complementary determining regions:
CDR 1 and CDR 3-> bind to complex MHC-antigen CDR 2->mainly binds to MHC
CDRs can shift position on antigen until tightest fit in formed

ii. V(D)J recombination-. Variable Joining and Diversity
*Same process-> variability in TCRs and immunoglobulins (like antibodies) from B-cells
*Each alpha and beta chain made of 2 or 3 segments
*Many alleles for each segment
*Segments recombined randomly
*Recombination between segments because of recombination sequences between segments and RAG proteins (Recombination Activation Genes)
*RAGs only expressed only in B and T cells

1. Alpha-chain pool
*42 V segments
*48 J segments
*2 D segments 1/42*1/48*1/2=1/4032

2. Beta-chain pool
*43 V segments
*12 J segments
*2 D segments

3. V-J vs V-D-J combinations
*Alpha and beta chains can be made of all 3 or just 2 segments

4. Promiscuous dimerization
*Whether dimmers form based on amino acid sequences of segments in respective alpha and beta chains

5. Junctional Diversity
*Within hypervariable regions, hypermutable DNA sequences especially true regions that connect VD and J segments.
*Specific proteins act as these sites to cause random points of mutation

c. T-cell receptors
*1013 positive T-cell receptors in one person. Is more than total number of T-cells in a person

i. Sensitivity of T-cell receptors
*Need around 10 MHCs presenting matching antigen to TCR
*Any given mature dendritic cell has >1,000,000 MHCs presenting different antigens at a time

d. Selection for T-cell receptors
*Strong interaction between T-cell receptor and MHC with antigen
->Clonal expansion of T-cell
->T-memory (same segments)

i. Positive selection
*Thymus- selects for TCRs that: strongly bind to non-self antigen
*Weakly binds to self antigens

ii. Negative selection
*Vast majority of T-cells ever made are killed in thymus

**April 10

I. Activation T-cells
a. Co-receptors
*Strengthen interaction between T-cell and dendritic cell
*Induce signal transduction
->Activation of T-cell

i. CD4 and CD8
*Interact directly with MHC

ii. CD28 and B7
*CD28 on T-cells
*B7 expressed by mature activated dendritic cells
*CD28 binding by B7 signal transduction and activation of T-cells

1. NF-kappa-B signaling
*Activated by TCR-MHC interaction and by CD28-B7 binding

b. IL-2
*NF-kappa B activates transcription and translation of IL-2 and IL-2 receptor
*IL-2 receptor is the major growth factor for T-cells
*In clonal expansion IL-2 stimulates T-cell division

c. Homing to secondary lymphoid tissue
*CCR7 on activated T-cell surface interacts with CCL2 on lymph node endothelial cells
*This allows T-cell diapedesis thru lymph node venule to get to interior of lymph node

d. Proliferation of activated T-cells
*CD4+ T-cells multiply to around 10,000 times starting number in 24 hours after activation
*CD8+ T-cells around 50,000
*CTLA4 (stops clonal expansion in T-cells) expression turned on around 24 hour after IL-2, IL-2R expression

**April 15

I. Differentiation of T-cell subtypes
*CD8+ T-cell development a result of interactions with CD4+ T-cells and IL-12
*All differentiation in T-cells (even irreversible changes) from change sin gene expression not change to DNA sequence of cell

II. CD4+ T-cells

a. TH 1
*Secrete IFN-gamma
*IFN gamma-> activation of NK cells
-CD8+cytotoxix T-cells
-Macrophages (induce B-cells to express pro-phagocytic antibodies<- opsonin Ig-G)
*Recruit phagocytes to deep tissues
*Ig-G kind of antibodies that can stimulate classical complement pathway

b. TH 2
*Secrete cytokines that
-Activate mast cells, basophils, eosinophils
-> Expulsive responses
->Release antimicrobial contents of granules
*Dominant in asthma
*Activate B-cells to release “barrier antibodies”-> IgE not good opsonin but cover microbe disable function of microbe

c. Follicular T-helper cells
*Interact with B-cells in secondary lymphoid tissue

d. TH 17
*Secrete IL-17 a cytokine which activates neutrophils attracts to site of inflammation

III. CD8+= cytotoxic T-cells
*Mechanisms of toxicity to intracellular pathogen infected cells
*Perforins, granzymes=cytotoxic chemicals stored in granules in CD8+ T-cell contents released on TCR-MHC I-CD 8+ binding, perforings granzymes lyse target cell
*Fas-L on T-cell interacts with Fas-R in target cell -> apoptosis
*CD8+ cell protects self from own cytotoxic chemicals by lining granules with cathepsins, which block perforin granzyme effects

IV. T-memory cells
*Naïve T-cells maintained by IL-7
*Over time ability to produce IL-7-R is reduced so fewer and fewer naïve T-cells over time
*Memory T-cells also maintained by IL-7
a. CD4+
*Maintained by IL-7 and antigen MHC-TCR interactions
*Survived for around 10 years

c. CD8+ memory T-cells
*Live around 30 years with out stimulation by antigen
*Maintained by IL-7
*Important for defense against very mutation prone viruses like flu
*Can go thru additional clonal expansion in response to renewed stimulation with antigen- this especially happens with latent viruses
*Latent viruses hang out in cells typically asymptomatic
*Overtime entire population of CD8+ T-memory cells just against latent viruses you’re infected with

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