Marion 5

Clonal Selection Hypothesis (see nature article)
Hypothesis developed by McFarland-Burnett. Attempt to satisfy all the observations about immune function at the time.
Erlich in 1890s and early 1900s studied humoral immunity. Some of his ideas were not far off. He had a term horror autotoxicus- term for autoimmunity. Immune system responds to foreign antigens, but response to self must be eliminated. Burnett’s explanation: (controversial- not first to come up with ideas)
For everything to work, diversity had to be preset in that:
1. Antigen selects for specific lymphocytes. Every lymphocyte has a unique antigen-specific receptor and sits around until it is activated.
2. Activation of a lymphocyte depends on antigen binding.
3. Every derivative of the activated lymphocyte is identical to the original.
These are obvious now, but not at the time.
4. Self-reactivity has to be eliminated.
Burnett included number 4 because he realized experimentally from Medawar’s data that self-nonself discrimination is made at birth. Medawar took cells from one strain of mouse and injected into neonate of another strain. Grew up to be tolerant to cells from injected strain of mouse as well as its own. This led him to generate the clonal selection nypothesis.
New paradigm: Janeway’s hypothesis:
self-tolerance and autoimmunity- maybe Burnett’s hypothesis does not apply anymore because of “danger signal” idea.
The job of APCs is to recognize patterns relevant to pathogens. Tolerance is about responding to nonself because self molecules do not activate APCs to activate T cells.
Another theory from Mel Cohn:
2-signal hypothesis: realization that antigen binding to receptor in most cases is not enough for activation. If a T cell binds to an APC with exactly the right peptide-MHC combination but the APC is not activated by danger signal, the T cell is made tolerant or shifted into anergy. During development, it would be killed outright. Anergy is important part of tolerance. Epithelial cells have MHC and can present, but cannot make danger signal molecules. Only the professional APCs can do that . B cells and T cells do get eliminated in development if they react to self. If they escape, get inactivated in periphery. If they are not inactivated, then autoimmunity develops.
Burnett’s theory is still applicable. Immune system in mice creates T cells over first 2 weeks of life. Birth- thymus is very large and produces lots of T cells. By 2-5 yrs in humans and 6 weeks in mice, thymus shrinks and output drops off. Bone marrow transplant- thymus enlarges again.Reason neonate is tolerant- during first week T cells learn difference between self and nonself. the mouse grows up tolerant.
Back to 2-signal hypothesis-
1. derived from receptor encountering antigen.
2. For T cell- danger signal from APC- costimulatory molecule. In its absence, theT cell is not activated.
For B cells- second signal in form of signal from T cells that B cells have to see for full activation. One class of B cells can get that signal from pathogens directly.

B cells are derived from SC-> CLP-> Pre-T or Pre-B (B-cell precursor s preferred term) or NK.
CLP kit interacts with stem cell factor on surface of bone marrow stromal cells. There has to be physical contact. Not clear how decision to become one or other is made. For B cell precursor, IL-7 has to be around. 2 transcription factors must be turned on for CLP to turn into B cell- Pax5 and E2a. Something turns those on- not clear.
ONce decision is made, becomes a pro-B cell. Most identifying characteristic is activation of Rag1 and Rag2 and initiation of rearrangement beginning at DJ locus. Once rearrangement of D and J is successful, you can get transcription, which is signal for early pro-B cell to attach V to DJ rearrangement. TdT is on in early and late proB to add diversity. In front of every V gene is a leader sequence exon. Upstream is a promoter. To get rearranged, must be activity at promoter. downstream is an enhancer between V genes and constant region. Successful VDJ rearrangement brings promoter close to enhancer. You get a lot of protein produced- VDJ with mu constant region. Cell becomes a large preB cell. Most important characteristic of large pre-B is it produces functional mu heavy chain. The heavy chain can be seen with fluorescent tagging in these cells. There is some on surface as well. Bip was thought to hold heavy chain in ER until light chain was formed, but this is not true. Explanation- heavy chain is expressed with a kind of light chain forming preB receptor. Kind of light chain is called lambda five with V-preB. (v-preB-lambda5). Proteins not covalently bound, binds H chain noncovalently. Does it bind to anything? unknowen. If there is no expression on cell surface, differentiation stops. Critical for continued progression of b cell to adult, fully functional B cell.
Large pre B becomes a small preB. Gene rearrangement commences at light chain locus. Starts in large, progresses in small. Rag on, TdT off. Successful VJ at kappa or lambda locus makes functional light chain protein and cell becomes immature B cell. Distinguishing feature is receptor on surface. Then in immature B cell, rearrangement is off.
ProB- rearrange H chain locus to get VDJ. PreB- rearrange light chain locus to get VJ. Then B cell with functional IgM receptor results. There has to be some feedback regulation of Rag1 and Rag2 to turn off. Not known.
If unsuccesssful, what happens?
proB
1. VDJ dysfunctional- goes to allele number two.
2. VDJ works- goes to preB. not- cell dies by apoptosis.
In mature B cells, in most cases there isa DJ rearrangement on both alleles. Once the cell gets successful VDJ, one is irrelevant.
In preB cell, have kappa 1 and 2, lambda 1 and 2 for the light chain. Mouse prefers kappa locus, human randomly selects. Mouse tries to make VJ on one allele. Successful-B.
Not- try K2, then lambdas one at a time. 4 chances. All fail- cell dies by apoptosis.
In this process, B cell will have property of allelic exclusion. This is unique for Ig locus and TcR locus. It means even though you have multiple alleles, a mature B cell only expresses one allele of heavy chain and one allele of light chain. Why is this important?
If a B cell expressed two different alleles, it might have 2 receptors, one to bind pathogen and one to bind self. Signal from pathogen would make it make both antibodies. The correct antibody must be made, so we do not want recognition of different pathogens on the same B cell to waste antibody.
Allelic exclusion- protein is expressed from only one allele- not like chromosomal inactivation.
Now we have immature B cell. Has a functional IgM on surface, but only IgM. Gets signal that antigen has been encountered. Reacts with Ag in bone marrow. Window of 2-4 hours (in vitro) after the IgM on surface develops. If the receptor binds something within this window of time, it is either clonally deleted by apoptosis or inactivated to become anergic (functionally inactive). Likely the only antigen it encounters here is self.
Degree of signalling determines inactivation or cell death. Experimentally- particulate/solid or cell surface antigen induces apoptosis. soluble antigens tend to induce anergy. Experiments were done with transgenic mice for antibodies to bind to MHC (apoptose) or DNA (anergic).
If B cell passes tolerance induction test, it becomes a mature B cell and moves to periphery, leaving bone marrow. Mature B cell has IgD and IgM. It goes out to occupy lymphoid tissue.
If immature B finds antigen in 2-4 hours of susceptibility to tolerance induction, it becomes tolerant or may be rescued by process of receptor editing. In immature B cell, RAG1 and RAG2 are off. If B cell encounters antigen, has chance to replace receptor with one that is not self-reactive. It initiates a new round of rearrangements at light chain locus in most cases. there are mult V genes and 5+J genes. If that light cchian is not self reactive, it goes on to maturity. Mature B cell with non-self reactive receptors IgM and IgD will become a B-2 cell or a B-1.
B-2 cells are ones we normally think about. normal antibody responses. they migrate to lymph nodes and spleen follicles, or Peyer’s patches. Sit for about 3 weeks. No antigen- they die. Not into follicle, die within a few days. What controls going into lymph node- HEV on venous capillary side inside lymph node. Have receptor expressing molecule CD62L. B cell has ligand to bind. Chemokines are chemmoattractants (SLC and BLC , secrete lymph chemokine and B lymphocyte chemokine.) These are secreted by follicular dendritic cells. These cells become memory cells. Responsive to T cell help. Can produce IgG, IgE, IgA. CD19hi, CD21low, IgMlow, IgDhigh (fluorescence results in flow cytometry)
B-1 cells are different. They go to area called marginal zone in spleen- area around follicle and PALS. Associate with metallophilic macrophages that are good at sampling blood into spleen. B-1 respond primarliy to bacterial carbohydrates. When activated, they produce IgM and usually do not undergo isotype switching. These also seem to have precursor homes to omentum. Characteristics of this precursor are not well known. CD19hi, CD21hi, IgMhi, IgDlo. (ffluorescence results in flow cytometry)
Rearrangement of Ig is an accident waiting to happen. Why? double-stranded breaks . first identified oncogene was myc2. Myc2 was identified in B cell lymphoma, in which an enormous number of tumors can be genereted from lymphoid cells because of intentional DSBs. If you have a cryptic recombination sequence in front that Rag 1 aand 2 can bind, and it induces a break and hooks it to wrong myc2 gene, you may place oncogene under one of most powerful promoters known. Gene puts cell in constant state of proliferation. Most of time works well.