Module 2 - Immunology

ELISA detects and quantifies

soluble proteins in biological fluids

Redox dye

Compounds that vary in their spectral absorbance properties depending on when they are in their oxidized or reduced state;

Used to determine presence of substance

Horse radish peroxidase

Redox dye which breaks down hydrogen peroxide & oxidizes itself — UV to visible spectrum

Direct ELISA procedure

1. Target Antigen is bound to the plate through adsorption and the rest of the plate is blocked with an inert protein

2. Enzyme-conjugated antibody is added and attaches to antigen

3. Redox dye is added, color forms in contact with molecule on the enzyme-conjugated antibody

Indirect ELISA procedure

1. Plate is coated with antigen and inert protein

2. Serum is added and target antibody binds to antigen

3. Secondary conjugated antibody is added to bind to target antibody

Quantitative (sandwich) ELISA

Uses a standard curve of pure target protein to determine the amount of protein in a sample

Capture antibody → Sample (antigen) → biotin-conjugated secondary antibody → conjugate for enzyme on secondary antibody → substrate solution

Immunohistochemistry is performed on

thin sections of tissues examined microscopically

Immunohistochemistry process

Primary antibody directed against the target protein is added then washed → secondary enzyme-conjugated antibody, wash → substrate producing color under microscopy

Trastuzumab (Herceptin)

Monoclonal antibody-based drug that targets HER-2 overexpressed in 1/3 of breast cancers;

Used as primary antibody in immunohistochemistry

Fluorescence

Physical property where an atom or molecule absorbs light at one wavelength and emits light at a lower wavelength

Emission wavelengths are always ___ than the absorption wavelengths

longer

Fluorescent microscopy analyzes

cells or tissues

Fluorescent microscopy process

Cells/tissues are fixed onto glass slides and stained with fluorochrome-labeled antibodies which are illuminated and viewed through a filter

Anti-nuclear Antibody (ANA) Test

Fluorescent microscopy test which detects antibody response (by addition of patient serum to grown human cells) against certain intra-nuclear proteins

Flow cytometry assesses

Expression of proteins at the surface of cells

Flow cytometry process

1. Antibodies conjugated with fluorescent substituents stain cells

2. Cells are passed through a cytometer (excites the fluorochromes and detectors sense light emitted)

Single-color flow cytometry data is displayed as a

Histogram

Dual-color flow cytometry data is displayed in a

dot plot

Western blotting

SDS-PAGE + antibodies to identify known proteins

SDS-PAGE

Separates a mixture of proteins on the basis of protein mass

SDS-PAGE Procedure

1. produce gel matrix (Acrylamide monomer + cross-linked polymer form a slab open on two ends)

2. Proteins extracted are solubilized in buffers with sodium dodecyl sulfate (SDS)

3. Proteins are injected into the gel and an electric current is supplied at the cathode

Sodium dodecyl sulfate (SDS)

Detergent that breaks up cells and regularizes the charge/mass ratio of the hydrophobic chains of the protein backbone

Beta-mercaptoethanol

Reducing agent sometimes used in SDS-PAGE to break up disulfide bonds in proteins

Coomassie Blue dye

General protein stain often used in SDS-PAGE

Western blotting procedure

1. Perform SDS-PAGE

2. Finished gel is placed atop buffer pre-wetted blotting paper + Nitrocellulose paper + more pre-wetted blotting paper

3. Electric current is ran through the gel so that proteins leave and are absorbed onto the nitrocellulose paper

4. Nitrocellulose paper is incubated with a series of antibodies (Primary for antigen and secondary enzyme-linked) then enzyme substrate for color or light production

T Cell Receptor structure

Composed of 2 polypeptide chains (alpha and beta) with n-terminal variable regions

Has a middle constant region and a c-terminal transmembrane region with a short cytoplasmic tail

T Cell Receptor variability is generated by

Somatic recombination

After rearrangement, TCRs do NOT

Change (no somatic hyper-mutation, affinity mutation, or equivalent of B cell “class switching”)

Complementarity determining regions (CDRs)

Hypervariable regions exist as loops at the far end of the receptor

TCRs have how many CDRs

3

The variable region of the alpha chain TCR is composed of

One variable and one joining segment via somatic recombination

The variable region of the beta chain TCR is composed of

A V, D, and J segments

Candida albicans

Microorganism that causes a benign yeast infection in women

Infection of the oral mucosa in the immunocompromised leads to Thrush

Severe combined immunodeficiency disorder (SCID)

Lack of RAG function (essential for gene re-arrangement) leading to the inability to resist infection

CD3

6 polypeptide chains which allow the TCR to leave the ER, transit the Golgi, and wind up on the cell surface

TCR complex

The TCR heterodimer associated with the 6 CD3 chains

Gamma-delta TCRs

Congregate at gut mucosal

Have much less diversity in receptors

Recognize lipid antigens and heat shock proteins (HSPs) in context of non-classical MHC molecules or no MHC

Class I pathway

Proteasomes degrade proteins and release short peptides or single amino acids that are transported into the ER to the MHC by IFNs and TAP. Degraded proteins meet up with MHC Class I proteins in ER and peptide-loaded MHC molecules are transported in vesicles to the surface of the cell

Proteasome

Multisubunit multicatalytic protease complex arranged into a stack of 4 rings with 7 subunits each

IFNs that use subunits to favor peptide transport into ER

LMP2, LMP7, and MECL-1

LMP2, LMP7, and MECL-1

Displace 3 corresponding proteasome subunits and substitutes for the main the structure; alters the enzymatic specificity of proteolysis by increasing cleavage after hydrophobic amino acids and decreasing cleavage after acidic amino acids

Immunoproteasome

the IFN-altered enzyme complex

IFN-γ initiates the production of

PA28 (proteasome activator)

PA28

Interacts with the alpha rings forming the entrance and exit portals causing a conformational change to open it wide (more proteins are processed and ejected before being totally degraded)

TAP (transporter of antigenic peptides)

Dimeric structure; each subunit contains a hydrophobic transmembrane domain that forms a channel and a cytosol-oriented ATP binding cassette (ABC) domain

TAP complex function

Hyrdolyzes ATP and uses the energy to translocate proteasome-produced peptides from the cytoplasm and into the ER

MHC Class I heavy chains in the ER are found

in a partially-folded state, complexed with calnexin

Calnexin

Chaperone protein that holds MHC Class I molecules in the ER and prevents transit into the golgi

Calnexin is displaced by

B3 microglobulin association with Class I heavy chain

Partially-folded alpha:Beta2 heterodimer associated with

calreticulin and Tapasin

Tapasin function

Forms a bridge with TAP complex

ERp57 and protein disulfide isomerase (PDI)

Help break and reform disulfide bonds in the MHC, facilitating peptide binding and the correct MHC folding

Once peptide is bound to MHC it

Finishes folding and the peptide binding complex dissociates

Class II pathway

Proteins are taken into the cell by phagocytic/endocytic vesicles where they are broken down; MHC molecules are taken out of the ER and fuse with phagolysosomes where they bind to the degraded protein fragments and taken to the cell surface

The acid protease activated by low pH which reduces disulfide bonds in a phagocytic/endocytic vesicle is

Cathepsin

IFN-lambda-induced thiol reductase (GILT)

facilitates reduction of disulfide bonds that aids in proteolysis in a phagocytic/endocytic vesicle

Invariant chain (Ii)

Chain which binds to Class II MHC to prevent internally-generated peptides from being picked up while in transit to the ER

Ii is cleaved into

CLIP (Class II-associated invariant chain peptide) — place-holder peptide

Ii:MHC complex is formed in the

ER

Ii:MHC complex must be transported to the _____ to bind with the vesicles fused with phagolysosomes

cytoplasm

HLA-DM

Associates with MHC Class II:CLIP complex to induce release of CLIP and allow cathepsin-degraded protein fragments to bind

The only cells that can sensitize naive T cells to antigen are

DCs

Antigen cross-presentation

the process by which externally-acquired antigen enters the Class I processing pathway

Immune response genes (Ir)

Genes responsible for the control over the ability to respond to a given antigen

The region encoding histocompatible genes (MHC Class I & II molecules) in mice is

H-2

MHC Class I loci in mice

K, D, and L

What is not encoded in the H-2 region (mice AND humans)?

Beta-2 microglobulin

MHC Class II loci in mice are

I-A and I-E

Major histocompatibility complex

H-2 region; contains genes for MHC Class I and II proteins, tapasin, LMP2, LMP7, and TAP

MHC molecules are passed down in genetics as a

Haplotype; one from mother and one from father

MHC genes are expressed

Co-dominantly

Human Class I loci

HLA-A, HLA-B, and HLA-C

Human Class II loci

HLA-DR, HLA-DQ, and HLA-DP

Polymorphism

Many gene variants in the population

Polygeny

Presence of several genes with similar function present within a single individual (several loci)

Who is likely to be a tissue/organ donor

The sibling (25% chance of the same two haplotypes)

For a peptide to strongly interact with the peptide binding groove, there must be. . .

Points of complementarity in shape, charge, and hydrophobicity between the peptide and corresponding contact points

Peptides that bind to MHC alleles display what AAs at anchor positions

hydrophobic residues

Variability occurs in the ______________ domains of Class I MHC

alpha 1 and 2

Variability occurs in the ______ chain in Class II HLA-DR

beta

MHC Genes are… (diversity wise)

Polymorphic and polygenic

Superantigen

Antigens made by bacteria or viruses that bind to common determinants on both non-polymorphic regions of MHC Class II molecules and TCR regions and activate the T cells that do not traditionally recognize the antigen

Staphylococcus aureus produces the superantigen

TSST-1

Exposure to superantigens can lead to

Toxic Shock Syndrome

Toxic shock syndrome Sx

Fever, malaise, low BP, vomiting, rash, multiple organ failure, and death

Superantigens induce the

Massive release of pro-inflammatory cytokines

Prolonged exposure of pro-inflammatory cytokines leads to the induction of

IL-10 (anti-inflammatory) and down-regulates MHC expression

Polymorphisms of MHC are restricted to the

peptide-binding “floor” or edges of helices facing the groove

How is T cell development similar to B cell development

Developmental stages are defined by receptor re-arrangements

Positive and negative selection mechanisms assure receptor quality and guard against auto-reactive cells

T cells leave the ________ to rearrange TCR in the ______

bone marrow; thymus

MHC Class II restricted T lymphocytes are

CD4 cells (turn into helper)

MHC Class I restricted T lymphocytes are

CD8 cells (turn into cytotoxic)

Thymus

primary lymphoid organ located in the anterior chest cavity above the heart and just below the notch of the sternum

Critical cells of the thymus

Thymocytes and thymic stroma

Thymocytes

Bone-marrow-derived T cell precursors

Thymic stroma

Network of epithelial cells tasked for T cell development

Lobules

Separate compartments in the thymus bounded by a capsule; contains the cortex and medulla

Cortex

outer region more closely packed with cells

Medulla

Innermost region of a capsule that is less dense with cells

T cell progenitors enter the thymus at the. . . and then divide and migrate into the. . .

Interface of the cortex and medulla;

Subcapsular region of the cortex