Immunology

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Class II presentation

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Done by Accessory cells:
1. Macrophages: (Monocytes) Phagocytose particles as well as live organisms.
2. B Cells: take up protein primarily thorugh Ig complexes and compement receptosr on surface. Very efficient since the membrane Ig can bind antigen with very high affinity thus low quantities of antigen required for response.
3. Dendritic cells, Langerhans cells (in skin) and Lymphoid dendritic cells: take up antigen, process it and can migrate to lymph node for activation of resident T cells.

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Pathways for antigen presentation by accesory cells

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1. Bind and internalize extracellular protein.
2. Movement of antigen into endosome, merge with lysosome, low pH and presence of proteases.
3.Fusion of peptide-bearing lysosomes with maturing classII chains -possessing vesicles from Golgi. Cleft created at this time by immediately filled by the free peptide.
4. the class II/ peptide conjugate then make it to the cell surface where it resides 12 or so hrs. before being recycled.
5. On the cell surface, the class II/peptide complex is recognized only by antigen specific T cells.

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Class I peptide presentation

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Vitually all cells express high levels of class I proteins.

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Pathway for peptide presentation via Class I proteins

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1. Proteins (self and viral) are sythesized in cytoplasm, degraded into peptides and pumped into RER. Proteosome cleaves the protein. Transport via specific transporters on the RER membrame.
2.Class I chain synthesized in RER. Class I chain binds the peptide and the beta 2 microglobulin protein - form stable structure. Mutant cells (lack either peptide or B-2 microglo - no class I protein on cell surface.
3. Ternary complex transported to surface where it has a life time of 10-15 hrs.

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Commonalities between class I/II presentation

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1. Both rely upon the positioning of a peptide in the cleft.
2. Both present T cells linear peptide sequences, not folds.
3. the polymorphic nature of the cleft gives rise to varied peptide binding.

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Antigen Presentation

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Refers to the presentation of peptides within class I/II clefts to TCR. To be expressed on the cell surface, all class i/II chains must have peptides in cleft.

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Why do class I/II proteins have peptides in their clefts?

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Because both extracellular (bacteria) and intracellular (viral) require T cell presentation.
1. T cells only recognize peptides.
2. T cells only recognize linear sequences, not folds in proteins like antibodies do.
3. T cells can only recognize the peptide in the context (presented by either) a class I or II protein.

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Major Histocompatibility Complex

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1. MHC gene products are found in the cell surface
2. The MHC producess two primary types of chains: Class I and II - control graft regection and immune response.
3. Both sets of proteins encoded are polymorphic. (most polymorphic region of the genome)

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Class I Proteins

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1. 3 distinct but highly related gene products, HLA-A,B,C.
2. Expressed as heterodimer with another protein, B2 microglobulin, which is not polymorphic.
3. Expressed co-dominant (A=B=C) on all cell types. Thus most people possess 6 diff. class I chains on every cell surface.
4. Possess 3 extracellular domains, transmembrane and cytoplasmic domains. Greater polymorphism in first 2, 3rd required for B2 binding. Also concerved for CD8 binding.

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Peptide cleft

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The most external 2 domains: hold 9 aminoacid peptides.
1. The sequence of the cleft defines tthe structure of the peptide bound.
2. Vistually all polymorphisms of class I map to this cleft, thus population is very polymorphic for peptides to be bound.
3. Class I chains are recognized by T cell receptors: both peptide and class I chain. Class I chains recognized by CD8 T cells, thus cytotoxic.

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Class II Proteins

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1. Heterodimer of and alfa and beta chain. Both are encoded within the MHC, both are polymorphic.
2. Sililar structure as class I chain with transmembrane, cytoplasmic tail, and extracellular domains.
3. Cleft that fits 9-15 aa peptide. Most polymorphic residues map to the cleft.
4. Expression of class II chain in association woth non-polymorphic chain, invariant, which acts as chaperone to cell surface.
5. Expression of class II chains is restricted B cells, macrophages, and dendritic cells. Cells important for antigen presentation.
6. Class II recognized by TCR (recognizes Class II and peptide via CD4 - thus helper cells).

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Primary response

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Is the response of such IgM/IgD B cell antigen the first time.
5-10 days
Relatively small response
IgM major product
Low affinity of antibody for antigen
All immunogens can work (protein, carbs, nucleic acid, etc.)
Requires high dose of antigen

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Secondary response

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Is the same for all succeeding exposures to antigen (n>1)
1-3 day lag
Relatively large response
IgG, IgA or IgE products
High affinity of antibdy for antigen (reflects mutation within VDJ)
Only peptide immunogens work (thus requires antigen presentation pathway)
Can get optimal response with much less antigen that primary response requires.

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2 Ways to activate B cells

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B cell activation is required for for antibody secretion:
1. T cell (T cell dependant antigens)
2. Polyvalent antigen (T cell independant antigens)

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T cell dependant B activation

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1. Ligation of cell surface antibody with the antigen
2. T cell help via the release of cytokines.
3. CD40 ligation via CD40 ligand expressed by T cells

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T cell dependant B activation provides for 4 steps for the B cell to achieve:

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1. Proliferate/differentiate to IgM producing cell.
2. Isotype switch to produce other Ig forms
3. Increase antigen/antibody binding via affinity maturation pf Ig chain gene
4. Progress into memory or plasma end stage cell

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How does the correct C cell sees the correct B cell?

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1. B cell takes up antigen, via cell surface Ig, to which it is specific
2. Antigen gets cleaved, generating series of 9-15 aa peptide.
3. These peptides can then be found within the cleft of a class II chain on the surface B cell.
4. The T cell antigen receptor then recognizes this complex

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Accessory Molecule

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CD40 protein! serve to:
1. Increase the affinity between two cell types
2. Increase signal
transduction in such cells
3. B7-1, B7-2 expression as means of further identification

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Importance of Ligation of CD40

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Ligation of CD40 is required for the B cell to further differentiate.
CD40 expressed by most periferal B cells
CD40 ligand (gp) expressed on activated T cells
Human mutations in CD40 ligand: B cells produce IgM but do not demonstrate much isotype swithching.

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How do T cell cytokines influence B cell behavior?

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1. Proliferation: IL-2, IL-4, IL-5, IL-13 are known T cell products that clearly stimulate B cell proliferation.
2. Antibody secretion: The use of a transmembrane bound of soluble Ig. Influenced by cytokines. IL-2/IL-6 induce Ig secretion by B cells.
3. Isotype Switching: via chromosomal recombination - B cell is fixed to produce a single isotype of Ig.

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Affinity Maturation

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After B cell are activated and class switched a portion migrate to spleen and lymph nodes:
1. Rapid proliferation- every 6-12hrs=1division
2. B cells sustain somatic mutation at Ig chains:to create Ig with higher affinity for antigen, those B cells that lose affinity for antigen are eliminated.

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Memory and Memory cells

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Subset of B cells that do not cycle up, but instead cycle down. They turn off and enter an anergic stage. They hold there untill they are sensitive to stimulation all over again.

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T cell independant antigens: Natural antibodies

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Self stimulate B cells via repeated structural moiety. B cells possesses cell surface Ig that recognizes a repeating epitope: polysaccharides, nucleic acids, glycolipids. This results in the crossliking of many surface Ig chains at same time.
IgM isotypes, low affinity, poor quality. No secondary response.

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Differences in the ability to respond to different antiboies, two important historical facts:

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1. Any single individual produces 1X10^8 ab's.
2. Two distict regions:
constant-does not change
variable-varies even amongs immunoglobulins of the same subclass- some noted to be hypervariable.

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Two Models for the generation of diverse proteins sequence

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Somatic Mutation Model and
Recombination Model

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Somatic Mutation Model

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All B cell make an identical Ig protein chain but each individual line of B cells mutates the coding sequence to give rise to different Ig proteins. Thus mutation in the coding sequence are inserted during B cell maturation and differentiation.

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Primary mode of antibody production

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variable region combination upon constant region sequences
However, during affinity maturation the specificity of the Ig chain ca be altered via mutations within the variable region.

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3 families of immunoglobulines genes

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heavy chaine gene
light chain gene kappa
light chain gene landa
constant and variable regions
light chain is either kappa or lambda

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Monoclonal B cells

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for any given B cell, its product is a single Ig protein made up of 2 identical light chains and w identical heavy chain proteins

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Antibody generation

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Heavy chain contain VJD , its recombination precedes light chain rearrangement
Light chain contain VJ and can utilize either kappa or lambda genes

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Terminal transferase

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Insert DNA sequences that are not derived form either of the joining pair.
it adds single nucleotides processively at 3'end to generate even more diversity

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Class switch (generation of isotype): Only heavy chain gene

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The isotype is defined the invariable regions (C) down stream from VDJ.
Utilizes recombination to remove such constant region sequences so as to allow the VDJ sequence to be placed upstream of different C regions
The antigen specificity does not change only the type of antibody produced

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Allelic Exclusion

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A single B cell only produces a single Ig chain. only one functional heavy and one light chain ONLY

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T cells possess a cell surface receptor that:

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1. is MHC restricited. Only responsive antigen when presented by self MHC.
2. Is antigen specific. for single protein and/or single peptice

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T cell receptor characteristics

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1. made of 4 different proteins: alfa, beta, gamma, and delta. expressed as heterodimer of a/b and g/d
2. Any given T cell only expresses one heterodimer.
3. Structure of TCR proteins is very similar to that of immunoglobulin

All possess the VJ or VDJ similar to Ig chains
All possess C region domains including transmembrane domains
No isotype switching: C region remain constant
The delta and beta chains possess D regions so are potentially more diverse than the gamma or alpha proteins.

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T cell differentiation

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Early T cells possess the TCR genes in their germline configuration. As the T cells develp in the thymus they begin to rearrange their TCR chains.

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TCR complex of proteins

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* CD3 complex 3 chains known as gama, delta, and episolon.
* homodimers and TCR complex
* signal transducin zeta chain and nu chain
* Mature TCR as alpha/beta: gamma delta episilon zeta 2

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TCR activation

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When T cell comes in contact with antigen/MHC the cell distorts the TCR. Distortion causes CD3 chains to serve as an activation signal.

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Accessory Molecules

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* CD4 recognizes class II chain. Also receptor for HIV. Helper cell
* CD8 as heterodimer or homodimer. binds to class I, CTL.

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What happens after a T cell is activated in an antigen specific fasion?

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1. Proliferate: in an autocrine fashion. Via IL-2 and IL-4 (clonal expansion), generate memory cells.
2. Effector funcions: Helper T cells produce cytokines=inflamatory and humural response. CTL's killing cells

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T cell maturation in the thymus

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1. The most immature T cell do not possess CD3 ... TCR
2. First observed change, cells begin their TCR rearrangement. gamma-delta first, alfa-beta second.
3. TCR genes start rearranging, transcription of CD3 is stimulated CD3/TCR made together
4. CD4 and CD8 now expressed "double +"
5. cells choose to be CD4 or CD8

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Positive selection: 2 models

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Saved from death by positive selection:
Instructional model: the double + cell binds to class I or II MCH with TCR. If close enough to match TCR then either CD4 or 8 selected.
Stochastic model: double + randomly lose expresion of either CD4 or CD8 chain. then engages to calss I or II or it dies.

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Negative selection:

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removal of CD4 or CD8 that will recognize self MHC when occupied by selfantigen.
if fail to do so T cell clones can help B cells to produce auto reactive antibodies and make CTL that kill normal self cells.

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The TH1/TH2 paradigm

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TH0-Naive, unactivated T cells.
a) Naive cell activated by IL-4 comits to TH2
b) if activated by IL-12 commits to TH1

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TH1 and TH2

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TH1. produce IFN gama= antibacterial, inflamatory response, activation of macrophages, enhanced CTL, NK and neutrophil killing
TH2. primary product IL-4, IL-5, IL-10. allergic response, IL-5 eosinophil proliferation and activation, and macrophages are pushed into an anti-inflamatory status.

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Severe Combined Immunodeficiency (SCID)

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Both B and T cell immunity affected.

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Bruton's Agammaglobulemia

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Sex-linked only males
Bruton's tyrosine kinase encoded in the X chromosome (Xq21.3-22)Prevents B cell from becoming a mature B cell
Detected with flowcytometry
Severe sinopulmunary and GI infections at 4-6 months. Polio, meningoencephalitis, dermatomyositis, lymphomas.
Treatment w/ gammaglobulin infusions

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Hyper IgM (HIM) Syndrome

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Sex linked mainly males.
Block above IgM (and IgD) in the sequence of B cell develpment (Xq26.3-27)
Severe sinopulmunary infection, may develop hepatic cirrhosis, neutropenia.
Bone marrow or stem cell transplant. most treated w/ gammaglobulin

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Immunoglobulin A deficiency

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Asymtomatic in up to 2/3 of patients who might experience mild respiratory infections or an increase in allergies such as food allergy, eczena, or hayfever dur to lact of immune exclusion provided by IgA at mucosal barrier
No treatment

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Common Variable Immunodeficiency Disease

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May be related to IgA deficiency through common suceptibility gene in class III HLA region.
Cessation of good antibody production (IgG, IgA, IgM) w/onset or recurrent, bacterial sinopulmonary infections and diarrhea.

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Treatment of antibody deficiencies

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Intravenous or subcutaneous gammaglobulin

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Diagnosis of antibody deficiency

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based on quantitative determination of IgG, IgM, IgA, and IgG subclasses by nephelometry as well as immunofixation electrophoresis to rule out monoclonal gammapathy of unknown significance and myelomas in adults.
ELISA
Flowcytometry

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Cytokine types

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1. Lymphokines
2. Inerlukins colony stimulating factors
3. chemokines

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Properties of cytokines

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1. modulate the immune response
2. have short 1/2 life in serum
3. produced and act up on a variety of cells
4. redundant
5. influence other cytokines
6. recognized by specific receptors on target cells

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How do cytokine receptors signal

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receptors cluster on the membrane
this results in JAK
pathway if phosphorilation dependant
JAK activation recruits STAT's
STATs are phosphorilated and form dimers
Dimers move into the nucleous and bind to target genes and initiate transcription
Individuals deficient on JAK or STATs show predictable loss of cytokine response

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How does cytokines stimulues gets turned off?

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recruiting of competitive inhibitors
bring in phosphotases that remove the phosphate groups

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Cytokines that influence Innate Immunity

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TNF
IL-1
IL-6
INF alpha and beta
IL-10

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Adaptive Immunity Cytokines

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IL-2
IL-4
INF gamma
IL-12

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Innate Immunity:
TNF

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Includes cytokines that lead to cell activation and proliferaion and ohters that lead to cell death.
Most immediate response to bacterial infection, induced by LPS
Primary sources LPS activated macrophages and mast cells
1. recruits neutrophils, monocytes, lymphocytes.
2. up regulates the killing efficiency of neutrophils and eosinophils
3. Increase class I proteins
4. produce other cytokines
At high doses:
1. fever
2. production of IL-1 and IL-6
3. incresed phagocytosis
4. supress bone marrow cell from dividing

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Innate Immunity:
IL-1

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Produced by activated macrophages/monocytes via TNF, LPS and exogenous IL-1
Enhances localized inflamation response
Function inhibited by natural competitors.

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Innate Immunity
IL-6

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Require action of IL-1 and INF

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Innate Immunity:
IL-10

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produced and activated by T cells, B cells, monocytes, and keratinocytes.
CAN INHIBIT the production of a number of cytokines, including IL-2, IL-3, IFN-gamma, GM-CSF, and TNF

Very potent inhibitor of macrophage activation

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adaptive immunity
INF gamma

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produced by TH1 activated T cells
1. anti-viral and anti proliferative
2. activate macrophages to kill phagocytosed bac
3. up regulates NK and neutrophil function
4. shift THO cells to TH1 upon activation

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Adaptive Immunity
IL-12

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Highest producer of B cells and monocytes
1. strong activator of NK cells
2. CD4 + TH0 cells, commit to TH1 pathway
3. Increases activity, proliferation of CD8

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Chemokines:

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Recruit cells into sites. Important in inflamatory responses in organizing and forming lymphoid tissue.

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Colony stimulating Factors

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c-kit and ligand
IL-3
GM-CFS
Macrophages colony stimulating factor
Granulocyte CSF
IL-7

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colony stimulation factor
c-kit and ligand

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interaction required for stem cell maintenance and expansion

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colony stimulating factors

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broad activator of bone marrow expansion: prologed sposure produces mast cells
GM-CSF: neutrophil/macrohage development
Macrophage colony stumulating factor: helps in final production of macrophages
Granulocyte CSF: push cells to granulocyte lineage: neutrophil and eosinophils
IL-7: B cell/T cell growth factor

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properties of the innate immunity

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* initial response to microbes, limiting or preventing infection
* adaptive immune responses use the effector mechamisms of the innate response
* innate response stimulates adaptive responses, and influences the nature of adaptive response

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Components of the Innate immune system: Epithelial barriers

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*Defensins: cystein rich peptodes with 3 disulfide bonds
*Barrier epithelial and serosal cavities contain intraepithelial T lymphocytes and B1 B cells

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Phagocytes: Neutrophils, and Macrophages

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*PMNs, monocytes, and tissue macrophages are derived from bone marrow precursors and circulate in the blood.
* PMNs are short lived are the first at site of inflamation
* Monocyte/macrophages live for much longer periods
* Profesional phagocytes cells function to destroy invading pathogens and possess specialized granules and molecules for this purpose.

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Recruitment of Phagocytes

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*Chemokines activated cells, induce receptors necesary for homing and transmigration through endothelium, and establish gradients sensed by cells
* f-met-leu-phe, C5a
*Defensins, lipid mediators
* CXC & CC chemokines

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fMLP

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7 transmembrane spanning receptor, linked to G protein signaling
Results in rapid recruitment to sites of infection.

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C5a

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*complement serum proteins that are sequencially activated by microbes resulting in generation of active anti-microbial polypeptides.
* the terminal peptide is a potent chemoattractant for phagocytic cells.

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Multistep Model of Leukocyte Recruitment

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* rolling of leukocytes on endothelium
* activation of leukocytes
* stable adherance of leukocytes to endothelium
* transmigration of leukocytes through the vessel wall

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How are microbes recognized as foreing by the innate immune defenses?

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*they possess unique structures that interact wiht receptors for pahtogen associated molecular patterns (PAMPS)
* Microbial products directly trigger inflamatory responses in cells of the innate defenses resulting in the porduction of cytokines

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NK cells in the innate response

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Important response against virusesand some intracellular pathogens.
Receptors are not specific for particular pathogens, but do allow discrimination of infected cells from uninfected cells