More on Generation of Antigen Receptors (VDJ), Antigen Presentation on T cells

hypervariable regions

HVRs on CDRs

CDRs come from the junctions between VDJ gene segments

B cell VDJ

B cells pick 1 V, D, and J segment each

• 30 ish V segments (depends on the gene)

• 23 D segments for heavy chain

• 5 average of J segments

• 1-9 constant segments to choose from depending on the chain gene

they possess a specialized enzyme system to cut at their own DNA to isolate segments - create double-stranded breaks

the heavy and light chains recombine independently

segments are cut and shoved together to make a single gene (gene composed of a single exon)

antibody tetramer is expressed on the surface of immature B lymphocytes

to increase diversity, there are two genes for the light chain for B cells to choose from (kappa and lambda)

• once one gene is selected, the other is inactivated

end up with millions (2×10^6) of possible combos from all their variations, but there are more steps to increase diversity later on

main initial drivers of the VDJ repertoire

1. which VDJ segments are combined during lymphocyte development

2. which combo of heavy and light (2 possibilities) chain genes are paired together (less important than VDJ)

the antibody constant region

both light and heavy chains have a constant region, but it is just the constant regions of the heavy chain that make up the constant region of the antibody (Fc) as a whole

different chain genes

each gene contains different clusters of VDJ and C segments separated by gaps of varying sizes

VDJ gene segments rearrangement

overall process called VDJ recombination

guided by flanking DNA sequences - RSS - recombination guides

• form pairs of 12s to 23s

V connects to J and so on

cannot connect 2 of the same segment type

some segments face forward and others backwards, making for different structure making depending on the interaction

• each pair type loops differently to accommodate splicing - makes sure all pair types end up going the right direction

  • forward facing first segment - hairpin

  • reverse-oriented first segment - coil loop

• pair type also determines how the double-stranded break is fixed

with the forward-oriented hairpin, the rest of the segments are spliced out are let free as a sort of plasmid and are forever lost from the genome - coding joint

for the reverse, the coil region remains after splicing - signal joint

both joints, once complete, are ready for transcription

chosen VDJ segment recombination

RAG 1/2 (dimer) - lymphocyte specific endonuclease that initiates recombination

• RAG - Recombination Activating Gene

• makes DS breaks then other machinery comes to repair them

ligation and DNA repair - ubiquitous DNA repair machinery

• repair DNA via non-homologous end-joining

• other ubiquitous elements first bind and stabilize the DNA before ligase starts repairing

  • Ku and Artemis, bind DNA break and open up the DNA hairpins, allowing repair

• there is also enzyme tdt, which adds additional nontemplate nucleotides to create an overhang to ligase can actually bind to the fragment ends to start fixing

  • tdt - terminal deoxynucleotide transferase, special polymerase that adds random nucleotides - additional junctional diversity

  • adding contemplate nucleotides means the nucleotides added in the gaps are random, which is the extra variability that ends up becoming the HVRs (CDRs)

signal joint repair

from reverse-facing join

has precise junctions - can be directly ligated - no tdt, no extra random variability!

coding joint break is 5’ to 3’ so both ends need tdt overhang for ligase binding

full diversity of immunoglobin repertoire

1. unique heavy/light chain gene pairings

2. VDJ segment selection

3. variable addition and subtraction of nucleotides at segment junctions - tdt junctional diversity

all of this together generates approx 10^13 possible BCRs

TCR generation

only one gene for each chain (alpha and beta), so no extra variability there

• to make up for this, each gene has more V and J segments to choose from than in the BCR genes

• more segments mean that TCRs have more diversity than BCRs just accounting for these variability sources - this is the extent of TCR variability but BCRs go further (somatic hypermutation)

similar VDJ arrangement

use the same cutting enzymes as B cells, so the RSS and 12/23 rule also apply here

the alpha chain is analogous to the BCR light chain (so only has V and J segments)

beta chain analogous to the BCR heavy chain (has the D segments as well as V and J)

BCR constant region diversity

5 antibody isotypes (classes) - M, D, then G, E, and A

isotypes determined by C region, which determines antibody function

the distribution of each class’s functions is unique

M is huge - can form a pentamer, better for target attachment, 1 of the first antibodies to be expressed, not great affinity initially

E only for allergies/parasites (mostly found in mast cells and basophils)

D has no affinities? the other antibody to be expressed first with M - not ever really secreted, scientists don’t really know what it does

initial antibody goal

the first thing antibodies are made for is to more strongly activate complement to see if this is enough to resolve the infection (conservation of resources)

first antibody production

M and D are expressed first - alternative splicing dictates which one

• first 2 exons following the J segment group

• both co-expressed by default on the surface of immature B cells as receptors

other isotypes require isotype switching

from membrane bound to secreted

alternative splicing swaps between carboxy termini for transmembrane M (M1 and 2) and secreted M (SC)

bound antibody drives intracellular signaling, which is what initially activated the B cell

• once activated, the B cell begins clonal expansion and starts secreting antibodies (mature B cell)

expression of antibody classes besides M and D requires sequential deletion of C segments and is irreversible

antibody testing

can test for antibody isotypes as well as specific antibodies (latter can be used to diagnose infections)

the transition between transmembrane and secreted antibodies can be reversed

BCR and TCR epitopes

BCR - not just proteins but also lipids, carbs, etc., recognize the native conformation of target molecules, recognize parts of whole and intact target via epitopes

TCR - polypeptide epitopes only, must be processed and presented on MHC

theoretically have a MHC for every peptide that needs presenting

MHC recognition pathways

two major pathways:

1. intracellular (cytoplasmic) pathogens (e.g. virus) - MHC I - CD8 T cells = cell death

2. extracellular (or endosomal) pathogens (e.g. some bacteria) - MHC II - CD4 T cells = activation of direct kill methods (bacteria and parasites) or activation of B cells (bacteria/toxins)

MHC I and II

Class I - endogenous sensing, expressed on all nucleated cells, present antigens from cytosol (e.g. viral, self, etc.), present to CD8 Ts

Class II - exogenous sensing, expression restricted to “professional” antigen presenting cells (APCs), present antigens from extracellular space that have been engulfed via endocytosis/phagocytosis, present antigens to CD4 Ts (helper Ts)

MHC posession

all cells express MHC I because they need to be able to show that they are infected by viruses when invaded so they can be destroyed by killer Ts

• virus spike proteins get deposited onto the surface of the infected cells from when the viral particle membrane merged with the cell membrane to release its contents into the cell

only professional antigen-presenting cells have MHC II because they are the cells that can talk to helper Ts to get the adaptive immune system up and running

cytosolic peptide presentation

MHC I to CD8

1. virus infects a cell

2. viral proteins are synthesized in the cytosol via ribosomes

3. peptide fragments of viral proteins are bound by MHC I in the ER lumen

4. bound peptides are transported by MHC I to the cell surface

5. cytotoxic T cell recognizes viral peptide-MHC I complex and kills the infected cell

overview of endosomal peptide presentation

MHC II to CD4

some pathogens can survive after being endocytosed (some even prefer it), leading to the infection of the macrophage

• bacteria holed up like this can then persist and create long-term infections

the infected macrophage will present bacterial peptides on MHC II, calling helper T cells, which will communicate with the macrophage and “awaken” it from the disabled state the specialized bacteria put it in - the macrophage then unleashes its full phagosome arsenal (which was previously downregulated by the bacteria), killing the bacteria

sometimes the bacteria blocking off the phagocytic machinery can also prevent adequate MHC peptide presentation, preventing the T helper assist

a cell can present multiple different peptides using its many MHCs at the same time

overview of extracellular peptide presentation on B cells

MCH II to CD4

B cells express early antibodies (membrane-bound)

these surface antibodies bind antigens and allow them to be endocytosed

antigens are prepped and presented on MHC II

helper T binds, which confirms that what the B cell has identified is a foreign antigen, gives the green light and the B cell activates

btw, all T cells in these overviews are mature and have already encountered the disease in question, hence why they can green-light activation

DCs

central role as bridge between innate and adaptive

key antigen presenting cells that are especially good at activating naive T cells

• express high levels of MHC I and II

• activate naive CD 4 and 8

• become more efficient at activation in the presence of danger signals (have not covered this)

DC activation keeps T cells specific to bacterial infection?

DCs pick up antigens via TLRs

how peptides get on the MHC

peptides generated from ubiquitinated proteins in the cytosol by the proteasome

• proteasome is a large protease complex that degrades cellular proteins - targets proteins tagged for destruction by ubiquitin

• present in all cells

• produce short peptide fragments

peptides from the cytosol are then transported into the ER and further processed before binding to MHC I

• TAP transporter allows the movement of peptides from the cytoplasm into the ER - TAP 1 and 2 dimer

newely synthesized MHC I are retained in the ER (membrane found, facing into lumen) until they bind a peptide

• MHC I actually stabilized by binding peptides

• until bound, stabilized by other proteins like calnexin and retained in ER

• large protein peptide complex positions MHC I next to TAP transporter

• cytosolic peptides are transported into the ER by TAP

• peptides are trimmed (by protease) and loaded onto MHC I

• loaded MHC I is then ready for transport to cell surface (vesicle?)

in absence of pathogenic peptides, will load self peptides for examination

DCs know they have something to express if a PRR gets tripped

class II peptide binding

MHC II are generated in acidified endocytic vesicles from proteins obtained through endocytosis, phagocytosis, and autophagy

• source of peptides is extracellular or endosomal (exogenous)

• proteolytic degradation by endosomal proteases

• vesicle containing broken-down peptides merges with vesicle containing MHC II for loading

activation of DCs by danger signals increases MHC II expression on cell surface

• MHC II molecules are normally degraded at a high rate

• TLR activation inhibits this degradation and results in high level of MHC II on the cell surface

• this system is specifically attuned to dangerous peptides, and so limits the surface presentation of safe self peptides (bound MHC IIs are held in vesicles until expressed)

exceptions

for the most part:

• extracellular peptides - MHC II and CD4

• intracellular peptides - MHC I and CD8

but sometimes you need to swap things:

• cross presentation - extracellular (exogenous) - MHC I

• autophagy - intracellular (endogenous) - MHC II

cross presentation

extracellular antigens on MHC I done by DCs

may take in portions of other infected cells to present a peptide from their cytosol

important for initial CD8 priming (cross-priming)

autophagy

pathways can deliver cytosolic antigens for MHC II presentation - self antigens, viral antigens

autophagy - process by which cytosolic material is enveloped in a specialized vesicle and targeted to endosomes

the major histocompatibility complex - MHC

histo = tissue

many proteins involved in antigen processing and presentation are encoded by genes within the MHC region

• MHC is a complex with many genes coding for each protein component

MHC is polygenic and polymorphic - allows for recognition of a great number of peptides

• polygenic - determined by multiple genes

• polymorphic - occurs in many different forms (alleles - differ widely between individuals)

match of MHCs determines transplant acceptance

HLA - human leukocyte antigen

3 class II molecules - HLA-DR, DP, and DQ (each has 2 genes, A and B, for the alpha and beta chains) (RPQ = rip chord, to remember)

• class II genes closely associated so they can be regulated together

3 class I molecules - HLA-A, B, and C

• associated with B microglobulin

reason why MHC is polygenic - many different genes contribute

genes corregulated with antigen flux?

for any one pathogen type (intra or extracellular) you essentially have 3 chances to bind it (3 receptors per MHC type)

• antigen residues determine which peptides an MHC can bind

MHC expression upregulated by cytokines:

• IFN gamma (type II) - produced by CD8 and 4 and NK cells

• IFN alpha, beta, and gamma - produced during antiviral response

more on MHC polymorphism

depending on the class of MHC I or II, there can be thousands of alleles for a particular gene

MHC I has more alleles than II

appears that there is substantially more variation in beta alleles v alpha alleles in MHC II genes

germ-line (inherited) variation

a single individual has 2 alleles per gene, and 3 different classes for each MHC, so 6 MHC class makeups total - lot of diversity

diversity much higher at population level - if you don’t have a receptor makeup to present a certain antigen, it doesn’t mean other lack this as well - a single disease has a much harder time incapacitating a population because of this

issue - some MHCs are linked to autoimmune issues

MHC evolution

gene duplication and conversion resulted in the evolution of many different MHC alleles

allelic variation in MHC molecules occurs predominantly within tge peptide-binding region (groove of the hotdog bun)

evolutionary pressure was development of microorganisms

some alleles more common than others

receptor distribution

BCRs and TCRs are clonally distributes

MHCs are not

variability in MCH peptide binding cleft

different allelic variants of an MHC molecule bind different peptides

the shape of the binding cleft restricts it to binding only certain peptides (a large variety, but still not universal binding)

the amino acids that stick into the grooves of the cleft are what determine binding affinity - anchor residues

population polymorphisms mainly pertain to differences in residue binding ability

MHC restriction and transplantation

TCRs recognize specific antigens AND specific MHC molecules

a transplant from someone with different MHCs won’t be recognized and will be rejected and killed quickly (histoincompatibility)

rejection occurs because there is a small but significant population of your T cells that recognize non-self MHCs - alloreactive T cells

• when binding, these cells don’t care about the antigen these non-self MHCs are presenting, but rather launch a fierce immune response due to the MHC itself

• this isn’t an intended function of the immune system but rather an artifact - the immune system didn’t evolve for transplant situations, which are outside its scope, but it was designed to seek out anything non-self, and other MHCs apply

• alloreactivity - the ability of T cells to respond to allelic polymorphisms in MHC molecules when encountering MHC-expressing cells from a genetically different individual.

unconventional T-cell subsets

the rules for antigen presentation discussed above apply to conventional alpha-beta T cells

there are other types of unconventional T cells which recognize other kinds of antigens, sometimes in the context of MHC-like molecules

• gamma-delta T cells - ligands unclear, some proteins

• NKT cells (natural killer T cells) - microbial lipids

• MAIT cells - microbial metabolites