• responds exclusively to protein antigens
• express an antigen specific receptor that clonally distributes cells like the B-cells do

Structure of T-cell receptor (TCR)

• one molecule has an alpha+alpha region; the other has beta+beta region
• heterodimers are linked by disulfide bonds and are glycoproteins
• contains hypervariable(CDR) regions where protein antigens bind
• antigen binds TCR only thru MHC

• not very well characterized
• structurally similar the the α/β TCR
• recognizes protein and non-protein antigens
• antigens do not have to be presented by MHC

1. TCR is rigid and only reembles the FAB fragment of BCR
2. TCR only recognizes protein antigens and in a strict way (via MHC)
3. TCR is membrane bound and never secreted
4. no class switching in TCR
5. no affinity maturation

• has receptor proper(TCR) + other molicules = CD3
• CD3+2 zeta chains+CD28 is involved in sig. trans.

[image]

• CD4, CD8, some adhesions molecules, some (-) regulation molecules

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1. co-stimulatory
2. sig. trans.
3. homing - co-receptors control entry of T-cells to tissues

1. naive T-cells encounter protein antigen via MHC
2. T-cells secrete cytokines
3. cytokines stimulate T-cell proliferation=clonal expansion
4. T-cells differentiate into memory and effector T-cells

• "displays" protein antigens to T-cells
• different from B-cells that recognize antigen on their own
• was recognized as a "complex"=grp of genes that are inherited together
• complex was originally found to code for cell surface molecules=influence an organisms ability to accept transplanted tissue
• every mammalian tissue has MHC
• in humans MHC is referred to as HLA complex(Human Leukocyte Antigen)

3 classes of MHC:

based on the region of the gene complex where they reside

• Class I
• Glycoprotiens; expresses on surface nearly all nucleated cells(nearly all cells can be APC)
• Class II
• Glycoproteins; expressed on "traditional" APCs(dendritic cells)
• Class III
• encodes for various secreted proteins(excomponents of the complement sys.)
• molecule assoc. with inflammation

• gene codes for a transmembrane protein called alpha or heavy chain
• there are extracellular domains=they are called α1 & α3
• expressed with MHC proper is a small polypeptide called Beta-2-microglobulin
• a cleft(groove) between alpha 1 & 2 is the peptide(antigen) presentation site

(the peptide presented by MHC is only part of the complete protein antigen)

• also has transmembrane and extracellular domains
• has α1&2, β1&2 domains
• also has extracellular cleft but is larger than MHC class I and can present larger peptides

[image]

Response of MHC to exogenous and endogenous

antigens: Exogenous(extracellular)

• MHC II involved
• antigens are taken into the APC by endocytosis
• antigens can be pathogens like viruses, bacteria, foreign proteins

1. antigen is internalized in an intracellular vesicle then vesicle fuses with an acidic lysosyme(contains enzymes that break down proteins)--at the same time the MHC is being put together in the ER
2. lysosomes intersect with golgi vesicle containing MHC---the protien is chaperoned(assists in protein folding and delivery) by CD74 and cleft by a protein called CLIP
3. MHC II-peptide combo is externalized on the Antigen Presenting Cell

1. proteosome breaks up protein products of infection
2. transporters(TAP-1, TAP-2) bring protein pieces to ER
3. inside ER small protein peices bind with MHC and β2 microglobulin-each is stabilized by proteins called tapasin and calreticulin
4. complex is transported to the APC membranes for presentation

• after presentation, sig. trans. takes place through the T-Cell Receptor and co-receptors
• sig. trans. stimulates the production of cytokines

1. TCR binds to a specific antigen
2. cascade of dephosphorylation/phosphorylation rxn
3. activation of intermediates(2nd messengers) in sig. trans.
4. intermediates activate enzymes
5. enzymes activate transcription factors
6. transcription factors stimulate transcription followed by translation to cytokines

• large group of proteins that mediate immunity and inflammation
• produced by activated T-cells and macrophages in the innate system

1. produced in response to antigen stimulation
2. act on same cell(autocrine) or nearby cell(paracrine)
3. one cytokine may have many actions (plietropism)
4. many cytokines may have same action (redundancy)

1. interleukins
2. interferons

• first kind to be produced by T-cells(1-2 hrs post activation and are produced by other immune cells)

Actions:

- promote survival, proliferation, and differentiation of T-cells

- activation of eosinophils

- involved in B-cell class switching to IgE

• cytokines that interfere with viral duplication in cells
• activate immune cells
• increase recognition of tumor cells for destruction
•  increase resistance to viral infection

• after infection, T-cells will increase 100,00 fold with a doubling time of about 6 hrs(in response to proliferating antigen)
• naive T's differentiate to effector T's which lead to production of cytokine(by CD4+ & CD8+ or T-cytotoxic cell which produce cytoxic protein)
• effectors appear 3-4 days post infection

T-helper "sub-types":

depends on which cytokine exposed to

T_H1 and T_H2

• differ in cytokines produced, receptor expressed, chemokine receptors expressed

T_H17

• subset of T's that produces interleukin 17 & 22
• contributes to defense against certain bacteria and inflammatory disorders

• are low MW cytokines
• stimulate leukocyte movement from blood to tissue

• a system of proteins found in serum and expressed on cell surfaces that interact with each other and other molecules of the immune system
• called "compliment" due to it refers to "complementing" or "assisting" with the mecrobial action of the antibodies

1. serum was found to have a "factor" that could kill bacteria
2. found that 2 factors existed

a. heat labile(sensitive)=compliment

b. heat resistant=antibodies

3. heat labile factor termed compliment

• compliment system generates many "effectors" of immunity
• maost components are made by liver(hepatocytes)
• some made by macrophages, GI and GU tract
• inactive forms=zymogens(are inactive until proteolytic enzyme cleavage)

-this cleavage starts the compliment cascade

1. lysis of bacteria, viruses, other cells
2. opsonization=promotion of phagocytosis
3. binding of compliment receptors on immune sys. cells triggering different effector functions

• letters
• number
•  names
• smaller fragment of component are designated a or b (a=lrgr;b=smaller)

• works with antibodies in innate sys.
• works with ag/ab complexes in adaptive

1. classical
2. lectin
3. alternative

• initiated by antigen-antibody(triggered by IgM, IgG bound to antigen like microbial surface) complex
• Fc regions become available to compliment proteins

- C1 binds to those Fc regions

- that binding initiates enzyme activity and cleaves other compliment proteins

• cleavage products become C3 convertase and C5 convertase

- C3 breaks up into C3a and C3b(products)

- C5 breaks up into C5a and C5b(products)

-C3a and C5b are anaphylatoxins

-C3b and C4b are opsonins

Ag-Ab-C1complex»»enzymatic rxn»»enz. rxn C3 convertase»»enz. rxn C5 convertase

• mediators of inflammation
• not directly involved in compliment cascade
• bring in neutrophils by acting as chemoattractants

• attach to microbe surfaces to promote phagocytosis
• receptors on immune cells detect opsonins
• opsonization enhances phagocytosis

see notes--http://www.profelis.org/amc/vorlesungen/gifs/MMPE_13IMM_163_02_eps.gif

• starts with binding of MBL(mannose binding lectin) to microbes---MBL is similar to C₁ and activates C₄
• subsequent steps are basically the same

With the Classic, Alt., Lectin pathways→→end event is generation of MAC:

• MAC induces osmotic lysis of cells
• C5b, C6, C7, C8, C9 form a complex which forms a large channel in a target cell
• enable ions, small molecules to diffuse freely
• cell cannot maintain osmotic stability→influx of H₂0 and loss of electrolytes

• attract neutrophils
• stimulate release of inflammatory products by various WBCs
• act on endothelial cells to promote diapedesis

1. compliment components are highly labile(have short 1/2 life so they must react quickly or will be destroyed)
2. there is also active regulation of the system by other proteins
3. distance-C3b is destroyed if it moves more than     40nm away from the place that it is made

Immunological techniques

• Agglutination
• Immunodiffusion
• Immunoelectrophoresis
• ELISA
• Radioimmuno Assay(RIA)
• Western Blots
• Flow Cytometry

• yes or no
• aggregation of particulates caused by combo of ag/ab
• ab are called agglutinins
• particles involved in agglutination=RBCs, bacteria, latex particles
• common in blood typing and detection of antigen or antibody

-relative amounts of antigen or antibody

2 types:

1. Radial(Mancini) method

2. Doubling(ouchterlony) method

• serum with antibody is diluted into agar
• antigen is applied to agar
• forms a ring after added together
• ring size is relative to conc.

• antigen and antibody are applied to opposite sides of agar plate
• diffuse towards each other and forms a precipitation line(thickness=amnt)

• ag mix is electrophorised in a gel
• ab is applied to gel, diffuses, and forms an arc=where ag/ab binding occurs

ELISA-Enzyme Linked Immunoabsorbant Assay

and

Radioimmunoassay

• antibody is conjugated with enzyme and reacts with substrate to produce a colored product
• see notes

- RIA is similar to ELISA but safer and cheaper

• detection of protein antigens
• electrophoretic gel separates proteins
• xfer proteins to a membrane
• incubated with an ab specific for protein of interest
• ab is tagged either radioactivity or chromogenically
• expose membrane to x-ray or chromotograph cassette and if protein is there a band forms

• analysis and/or separation of cells identified by flourescent ab or stains
• a laser stimulates flourescence and a detector sends the signal to a computer
• detects cells and can separate them

ex. ab is flouresced and attached to CD4+ cells and laser exites them and computer quantitates number of cells

• inbred strains of rats, mice, guinea pigs
• produce selective mating of litter mates for 20 gen.
• called syngenic=genetically identical(can use for study w/o problem of genetic div.)
• used for organ studies, they don't reject organs
• inbred mice, SCID mice, thymectomized or congenically athymic(nude) mice, transgenic mice, knockout mice

SCID mice

(severe combined immunodef. disease)

• B's and T's fail to develop
• can accept human hematopoetic stem cell transfusions
• used for the study of lymphocyte development
• also used to test vaccines(ex. HIV vaccine)

• cells of thymus are renewed artif. by stem cells
• fail to dev. T-cells
• mutation also affects hair=nude

• inject cloned gene into mouse embryo(resultant eggs are xfered to "pseudopregnant" female)
• the genes become incorporated into somatic and germline(can pass to future gen.) DNA
• ex. the gene can be turned on by supplementation

Nut. control:transgene→egg→surrogate→enters genome→turn on exp. with suppl. and study effects 

• uses "gene targeting" method-replace normal gene
• technique "silences" gene to find purpose of gene

• uses "microarrays"(aka gene chips)
• study expr. of 1000's of genes at one time
• see notes