Immunology Exam 2

immuno exam2

What is ELISA?

enzyme-linked immunosorbent assay

What does ELISA do?

\-detect and quantify soluble proteins in biological fluids

\-detect antibodies in serum directed against specific proteins

\-quantitative data

What does ELISA depend on?

depends upon the exquisite specificity of bonding between an antibody and an antigen

What is spectrophotometry?

a way to see the amount of light being absorbed and its proportionality to the amount of absorbing compound

What is horse radish peroxide?

\-an enzyme that will catalyze the break down of hydrogen peroxide and in so doing oxides the TMB molecule

\-helps to shift the spectral absorbance from ultraviolet to the visible spectrum

What is an essential part to any immunoassay?

conjugated antibodies

What is a conjugated antibody?

antibodies that ate permanently linked to other molecules using chemical covalent crosslinking agents

What is immunohistochemistry?

similar to ELISA, but it is performed directly on thin sections of tissues.

What is fluorescence?

\-a physical property whereby an atom or molecule absorbs light at one wavelength and emits light at a different, lower wavelength (emission wavelengths are always longer than the absorption)

\-each molecule has their unique absorption and emission spectra

What is fluorescent microscopy?

the simplest immuno-fluorescent technique

What is flow cytometry used for?

to asses the expression of proteins at the surface of cells

What is SDS-PAGE?

sodium dodecyl sulfate polyacrylamide gel electrophoresis

What does western blotting identify?

\-allows the identification of a known protein through the use of an antibody

\-allows you to see the molecular weight of the target the antibodies are binding to

\-used after SDS-PAGE

What is a T cell Receptor?

a membrane bound glycoprotein with a structure that closely resembles a single antigen-binding arm of Ig

What is a T cell receptor composed of?

2 different polypeptide chains (alpha and beta) with n-terminal variable regions that binds antigen

What are the variable regions of a T cell Receptor called?

complementarity determining regions (CDRs)

What are the three CDRs?

CDR1, CDR2, CDR3

What is the structure of the variable region of the TCR alpha chain?

\-similar to the light chain of Ig

\-composed of one V and one J segment

\-one alpha chain constant

What is the structure of the variable region of the TCR beta chain?

\-similar to heavy chain of Ig

\-composed of one V, D, and J segments

\-two beta chain constants

What is the T cell receptor complex?

\-alpha and beta chains cannot leave the ER

\-associated with 6 additional polypeptide chains to make CD3

\-two eta, two zeta, one delta, one gamma chain

\-the TCR heterodimer plus 6CD3 chains make up the TCR complex

How are alpha and beta T cell important?

makes up we over 90% of the T cells in the body

What is the other 10% of T cells made of?

\-one made up of gamma and one made up of delta

\-much less diverse than alpha:beta

Where do the gamma and delta T cells congregate?

at gut mucosal sites

What do the gamma and delta T cells recognize?

\-lipid antigens and heat shock proteins (HSPs)

\-rapid responders for the gut

What is the proteasome?

\-multi-subunit, multi-catalytic protease complex arranged into a stack of 4 ring with 7 subunits

\-hollow core with enzymatic active sites and proteins are fed through either end for proteolytic degradation

What do proteasomes generate?

peptides for binding to Class I MHC

How does antigen processing via Class I pathway work?

\-interferons induce expression of 3 additional subunits

\-LMP2, LMP7, and Mecl-1 displace 3 corresponding proteasome subunits

\-substitutional alter enzymatic specificity of proteolysis, increasing cleaving after hydrophobic aa and decreasing cleaving after acid ones

\-changes favor peptide transport into ER and binding to MHC

What is the INF-altered enzyme complex called?

immunoproteasome

What does INF-gamma initiate production of?

4th inducible proteasome subunit called PA28 or the proteasome activator

What does PA28 interact with?

the alpha rings that form the entrance and exit portals for the intact proteins and their degraded proteins

What does PA28 do?

\-causes conformational change in the alpha ring, opening it wide

\-this enhances protein speed of entry/exit

How does the effect of PA28 help with MHC binding?

increases speed of entry/exit means more proteins are processed, but ate ejected before being degraded into amino acids which generate more material suitable for MHC binding

What is TAP?

-transporter of antigen peptides

What is the structure of TAP?

\-dimeric

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

What enhances the TAP complex?

interferons

What does the TAP complex do?

-hydrolyses ATP and uses the energy to translocate proteasome-produced peptides from the cytoplasm and into the endoplasmic reticulum

Antigen processing via the Class I pathway in simple words

\-proteins are degraded by the proteasome complex

\-the resulting short peptides are then transported by the TAP complex, in an ATP-dependent manner, into the lumen of the endoplasmic reticulum

synthesized MHC Class I proteins

What are the steps in Class I pathway?

\-appearance of MHC Class I molecule in the ER

\-formation of MHC Class I peptide-loading complex

\-loading the MHC class I molecule

\-transported to the surface of the cell to be recognized by killer cells

What is Calnexin?

\-chaperone protein

\-function is to hold the MHC class I molecule in the ER and prevent its transit into the golgi

What are calreticulin and tapasin used for?

for a bridge with TAP

What are ERp47 and protein disulfide isomerase used for?

\-help break and reform disulfide bonds in the MHC

\-facilitating peptide binding and of correct MHC folding

What does tapasin do?

keeps TAP and MHC in close proximity so that the transported peptides quickly associated with the binding groove on class I MHC

Once peptide loaded MHC molecules are on the surface of the cell, what are they recognized by?

CD8 cytotoxic T cells

What are the steps to antigen processing via the Class II pathway?

\-proteins are taken in via phagocytosis and subjected to pH drop

\-Invariant chain binds in the groove of MHC class II

\-Ii is cleaved to leave a fragment bound to molecule

\-short fragment (CLIP) is left as a place holder

\-transported to cell surface and presented

What does a pH drop do to the proteins?

\-reduced disulfide bonds in the proteins

\-makes them susceptible to proteolysis

\-fusion with lysosomes add acid proteases that are activated by low pH

What do cathepsins do?

digest the proteins into fragments that can then bind to the class II MHC

What is the most important acid proteases?

cathepsins (B, D, S, L)

What does INF-gamma-induced thiol reductase (GILT) do?

facilitated reduction of disulfide bonds that aids in proteolysis

What does HLA-DM do?

associated with MHC class II: CLIP complex, induces release of CLIP and allows cathepsin-degraded protein fragments to bind

What is antigen cross-presentation?

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

What are the two separate lines of evidence MHC grew originally out of?

\-tissue transplantation

\-vaccintation

What is tissue transplantation?

\-experiments using skin grafts of inbred mice

\-grafts transferred between the same inbred successfully engrafted while transfers between different strands failed

What did tissue transplantation help to make clear?

the mechanism of rejection was immune-related and tied to genetics

How was vaccination used?

\-vaccinated different strains with various antigens

\-for a given antigen some strains had a vigorous antibody response, while other had almost none at all

\-depended on both the antigen used and the strain of mouse

What did vaccination help to make clear?

that something in the genetics controlled the ability to respond to a given antigen

Why are putative genes called Immune Response genes (Ir)?

because immunization studies pointed to genetic control over the ability to respond to a given antigen

Why are putative genes known as histocompatibility genes?

because the other line of investigation showed apparent genetic control over tissue engraftment

How many class I loci that encode functional MHC proteins do humans have?

3

What are the 3 class I loci that encode functional MHC proteins?

HLA-A, HLA-B, HLA-C

Is beta2 macroglobulin encoded in MHC?

No

What chromosome is human MHC on?

chromosome 6

How many class II loci that encode function MHC proteins do humans have?

3

What are the 3 class II loci that encode functional MHC proteins?

HLA-DR, HLR-DQ, HLR-DP

What is the definition of functional in terms of this discussion?

defines as capable of presenting peptide antigen to a T cell

How is MHC diversity generated and maintained?

polymorphism and polygeny

Define polymorphism

many gene variants in the population

Define polygeny

presence of several genes with similar function present within a single individual

What do polymorphism and polygeny do?

ensure greatest variation both in population as a whole and individuals

Hoe are HLA alleles expressed?

co-dominant

How many combinations of haplotypes can be found in offspring?

4

What are the chances of two siblings having the same haplotypes?

25%

What MHC proteins present processes protein Ag to T cells?

HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR

Explain paleo-immunology circa early-mid 1980s

\-transplantation antigens=encoded MHC gene regions

\-many allelic variation, sequence differences mainly confined to limited regions

\-T cells from individuals with different MHC alleles recognized different peptides on the same protein

Explain one of the most important discovery in immunology

\-peptides bound MHC at special binding groove

\-groove perched atop stalk, facing T cells

\-groove closed at each end

\-polymorphism mostly restricted to peptide binding floor or edges of helices facing groove

What is the structure for Class I?

the α-helices surrounding the peptide-binding cleft bow inwards at the tips, essentially closing the cleft and limiting peptide size

What is the structure for Class II?

open at the ends, allowing longer peptides to spill over at each end

Where is variability most confines in human MHC?

to floor of peptide binding groove and to edges of alpha helices that come into contact with the processes peptide

Where does Class I MHC variability occur?

α1 and α2 domains

Where does Class II variability occur?

\-HLA-DR

\-Beta chain is polymorphic

\-alpha chain has little variability

the ends of peptides often have anchor residues that are \_______ in nature

hydrophobic

What do peptides that are bound to particular MHC alleles display?

common or similar amino acid residues at key anchor positions that mediates peptide interaction with MHC

How can a peptide strongly interact with peptide binding groove?

there must be points of complementary in shape, charge, and in particular hydrophobicity between the peptide and the corresponding contact points along the groove

What do T cell receptors recognize?

-the bimolecular complex of self MHC and peptide

What is allo-recognition?

the recognition of non-self MHC by T cells as though it were foreign antigen of microbial origin

What is MHC restriction?

A T cell is restricted to recognizing its peptide only when the peptide is presented on the specific MHC allele that positively selected the T cell.

What is the exception to MHC restriction?

allo-recognition

What cross-rection is the basis for tissue rejection?

1-5% of any individuals T-cells will cross react with non-self MHC

What are superantigens?

\-made by bacteria or virus

\-bind to common determinants on both non-polymorphic regions of MHC Class II molecules and non-variable regions of TCR

\-induce massive release of pro-inflammatory cytokines (TNF, INF-gamma, and even INF-alpha)

What is Staphylococcus aureus?

superantigen that can cause food poising and/or toxic shock syndrome

What does polyclonal T cell activation do?

activated useless cells, diverting resources from truly antigen-specific T cells that could combat infection

What can prolonged exposure to superantigens lead to?

\-induction of cytokine IL-10 which is anti-inflammatory and down regulates MHC expression

\-T cells are activates in a way to make them unresponsive to future stimulation

T cells are the

"brain" of the immune system

Where do B cells stay during their development?

in the bone marrow

Where do T cells develop?

leave the bone marrow, and rearrange in TCR in thymus

What is one division of T lymphocytes?

CD4 cells that are MHC class II restricted and the CD8 T cells that are MHC I restricted

What is the other major division of T lymphocytes?

between alpha beta and gamma delta

What is the thymus?

a primary lymphoid organ

Where is they thymus located?

in the anterior chest cavity above the heart, below the notch of the sternum

What types of cells does the thymus contain?

thymocytes and thymic stroma

What are thymocytes?

bone marrow derived T cell precursors