Marion 9 (Friday)

Humoral Immunity
2 types: innate and adaptive.
innate- soluble factors- talking about complement. It has been bane of immunology teachers and students because it involves lots of components. How proteins are activated can be complicated if you focus on details. He has provided notes that go through details. Think about the concept, what it does, why it is important.
Proteins constitute about 12 proteins involved in activation and about 6 or more involved in regulation and control.
Slide 1: 3 ways complement system engaged:
1. classical pathway
discovered by von Behring early 1900s. Erlich discovered antibodies, but von Behring and Kitasato realized antibodies were not enough to eliminate bacteria. Had to be something else. They called it complement, because it complements antibodies.
Cl path activated by immune complexes. Antibodies can neutralize viruses and toxins, and provide handles for phagocytes, but they need help.
2. lectin pathway-
most recent to be discovered. Involves protein in circulation that recognize mannans. Almost the only place to find exposed mannose is on pathogens, mostly bacteria. Not absolutely true. Humans- mannoses get glycosylated, mostly with sialic acid. Our cells only rarely show exposed mannose. There are a few nonsialated exceptions. All the mannoses on cell wall and capsule of bacteria are targets for protein to activate lectin pathway- mannan binding proteins (MBP), and have associated mannan binding protein associated serine proteases. This mechanism activates potent innate immune response dependent on series of complement proteins to recognize mannans.
2. alternative pathway
discovered in 1950s- classical was only one known at the time. Immune complexes were not only way to get activation.
works like lectin pathway, with differences. Depends on natural turnover of complement components. C3 hydrolysed to active C3b at low rate all the time. If it happens near a bacteria, can bind to surface. Then other proteins can use it as focus to activate more complement. Pathway becomes more important- once classical or lectin activated, alternative pathway allows for process of amplification for much more complement activation than in its absence. Components of this pathway are heat labile- why you heat inactivate fetal bovine serum before incubating cells in it.
Slide 2: Most important point about complement activation- generate something called C3 convertase (serine protease). It converts C3 to C3b +C3a and C3b binds to cell surface (mostly bacteria). C3a is lost to fluid phase, but it is an anaphylatoxin- induces inflammation
**important**1. cell- bound components are opsonins- "to prepare to eat". There are receptors for these components on phagocytic cells (PMNS, macrophages, phagocytic cells). Erythrocytes have them, too. Relevant for classical pathway- red cells are primary means for removal of immune complexes from blood to transfer to liver Kupfer cells- strip off complexes and get rid of them without damaging red cells.
2. soluble components induce inflammation.
What else can complement do?
Once you generate C3 convertase (cell bound), it is formed on cell surface. Converts C3 to C3b, binds more cells, etc.
Slide 3: C3 convertase, when you generate C3b, creates C5 convertase. Serine protease with different substrate, C5. Converts C5 into C5b, cell bound, and soluble C5a (anaphylatoxin- the most potent inflammation inducer from complement activation because of receptors on vascular endothelium to induce vascular dilation. Mast cells- histamine release. Smooth muscle- bronchial restriction).
4: C5b is on cell surface. C6(1),7(1),8(1),9(9-10) now polymerize around C5b to generate the MAC (membrane attack complex). Form pore to induce cell lysis. Looks like perforin complexes.
Complements cannot distinguish self from nonself surface. Complement activation is tightly regulated.
Soluble inhibition - keeps enzyme cascade in check. Once C3b is generated, get amplification because of alternative pathway.
cell surface inhibition- on our cells, not bacterial. Inhibits C3 convertase, inhibits binding of Clq to immune complexes.As burden of pathogen reduced, control mechanismss rein in system. Also inhibits MAC formation to protect our cells
See slides for review with diagrams from text.
Complement provides 3 means of effector function. For all 3 pathways, goal is to generate C3 convertase. C3b is ligand for handle for phagocytic cells**), Membrane complex lyses cell. Photomicrograph shows holes. Same structure as perforin. Sequence comparison: Structure C9 is similar to perforin.
Humoral Adaptive Immunity
Antibody (Ab)- selected during development of B cells to be good at binding pathogens. Generated by B cells. B cell has to be activated from naive inexperienced B cell to activated B cell, or plasmablast.
Plasmablast can go one of 3 directions:
1. memory B cell for later use
2. plasma cell- cell specialized for Ab secretion
3. die
2 types B cells:
Slide 6: B-1
Slide 5 and 7: B-2 are B cells that migrate to lymph node and spleen depending on expression of molecule on surface called CD62L. This molecule helps new B cell get into lymph node through HEV. HEV has receptors on lumenal side for this ligand. B cell goes to paracortex (T cell zone). Want to migrate to follicle. As B cell goes through, it interacts w T cells, + antigen brought in in lymph as well as dendritic and phagocytic cells. If the B cell interacts with a T cell and antigen to become activated, it forms a specialized structure called a germinal center (slides 10, 11). No contact, just goes into follicle and sits about 3 wks, recirculates. Lasts about 3 wks in follicle. Not in follicle the B cell lasts 3 days and dies. Samples all antigens from afferent lymph and with T cells interacts with APCs. In paracortex, or T cell zone (PALS) of spleen If B cell there and activated helper T cell, B cell found antigen it can bind and take up because receptors are crosslinked by antigen, binding to antigen delivers to B cell signal 1 through Ig receptor (aggregation of Ig receptor on surface is signal 1)
If antigen can be processed and peptides generated from it, recognized by T cell receptor (activated by dendritic cell, which had MHC+peptide and IL-2), cognate interaction happens.
If T cell did not have to see antigen on B cell, B with self and B with nonself would be equal.
Only way B cell helped by T cellis if B cell expresses same MHC peptide that activated T cell in the first place. Signal 2: For B cell to get help from T cell, needs signal 2. Signal 2 from T cell is in form of CD40 ligand or CD 154. Activated T cell expresses CD40 ligand. B cell has CD40 on surface. Molecule engages ligand on T cell for signal 2. B cell differentiate into memory or plasma cell in response to cytokines from Tcell, but signal 2 is essential for formation of germinal center.
If T cell does not see antigen but does see CD40, may see FAS and trigger apoptosis. signal 2 delivered by CD40 on activated T cell. When B cell gets signals in paracortex, migrates into follicle to generate germinal center, where clonal expansion (10^3-10^4 fold), affinity maturation (driven by T cells and dependent on CD40 ligand and cytokines including IL-4), and isotype switching. Also differentiation to plasma cell or memory cell. Signals for differentiation are unknown. Memory cells can sit and make antibodies for months to years.
Slide 12: Affinity maturation is due to somatic mutation plus selection. Selection more important. Ability of limited antigen to select B cells with best receptors to form memory cells is important. Follicular dendritic cells are not lymphoid derived. Have long dendrites all through follicle forming reticular network. They have on surface FCgamma and C3b receptors and derivatives. They bind immune complexes. They can hold immune complexes for long time- reservoir for B cell sampling. Antigen released from follicular cell to B cell, B cell expresses peptide in MHC, gets activated. May be one way long term memory is maintained. Read Gray paper.
Germinal centers – where above processes occur. B cell undergoes somatic mutation. In process generates dysfunctional receptor- dies by apoptosis. Self reactivity- dies by apoptosis. Why? because B cells have in germinal center as they proliferate in dark zone (where somatic mutation occurs)- move into light zone- proliferation slows down, but undergo test. Must be able to generate specific antigen in order to generate signal. Need receptor engagement on FDC and T cell help again – if not- dies by apoptosis. Failsafe mechanism maintains tolerance. B cell not relevant, it drops out. Passes- proliferates more, become plasma cell or sits in follicle as memory B cell.
Other points:
What about B-1 B cells?
These cells live in marginal zone of spleen and peritoneal cavity. They are selected for these areas because of specificity for bacterial carbohydrates. Activation is different. Signal 1 is same. Ig receptor. Signal 2 derives from antigen. Like innate immune B cell. Still activated in clonotypic fashion, but gets signal through TLR or other innate receptors. When cells are activated, limited proliferation (not much)occurs, very limited isotype switching, no somatic mutation, no affinity maturation, no memory.
Best Ab for bacteria are carbohydrates, but it is impossible to develop a strictly carbohydrate vaccine. Immune response is IgM and relatively low affinity antibody and no memory. Solution for haemophilus influenza- hook onto carrier to get T cell epitopes. B cell receptor complex other molecules important CD21,81,19
19 important because cytoplasmic tail binds cytoplasmic tyrosine kinases
21 complement receptor- binds immune complexes.
81 binds whole thing together.
Ig receptor comes together with CD19 to get more effective B cell activation than with B cell receptor alone.
In children, last immune development is response to carbohydrates- the lack increases susceptibility to infection.
See slides again for review.
IgA prevents colonization of surfaces.
IgE sits on surface of mast cells waiting for amtigen.
Secretory IgA- B cells in Peyer’s patch . Binds to poly Ig receptor, transported, released on lumenal side by cleavage of receptor, leaves secretory piece.
IgE found on mast cells lining epithelium. IgG throughout. IgM in circulation.
Look in his information he sent us.