Recognition, response and receptor organization. Flashcards
Describe and compare the terms affinity and avidity in the context of receptor-ligand binding in the immune system.
Since the role of the immune system is to handle an enormous variety of pathogens, there is need for an even bigger variety of receptor-ligand interactions. Affinity is defined as the collective strength of many weak non-covalent interactions between ligand and receptor. The high diversity of the adaptive immune system relies on receptors with extremely high affinity to specific ligands, but this takes time to produce enough off. During the early stages of the immune response, the concept of avidity is utilized. High avidity means that the collective strength of several low affinity bindings generate a strong bond despite the low affinity of a single receptor ligand interaction. For example, IgM is the first antibody class to be secreted during the immune response, and while the affinity to the target hasn’t been optimized, the ten binding sites on the IgM pentamer allows for high avidity.
Ligand-receptor binding induces
molecular changes in the receptor, which four types of changes can occur?
- Conformational changes
- Dimerisation (or polymerization)
- Location
- Covalent modification
Receptor changes often induce cascades of intracellular events leading to for example activation of enzymes or changes in intracellular locations of
molecules.
Give one example of how cells can calibrate the strength of a signal.
One example of signal strength calibration is the use of several receptor chains, which alone have low affinity and together higher affinity. This also allows for more different receptors from the same amount of genetic material. This is used for the IL-2 receptor which has three chains (three possible conformations): The alpha chain has low affinity while the beta-gamma chains have intermediate affinity. When they all come together the receptor have a high affinity to IL-2.
So, aggregation due to ligand binding can enhance ligand binding. This is often used to maintain cell-cell contact when needed over an extended period of time - like in the immunological synapse.
What is the advantages of the formation of immunological synapses?
The formation of immunological synapses allows for strong but highly localized signalling between cells. This is crucial in the immune system as you only want to activate just the cells that are needed and not something else.
What is the difference between antibodies and BCRs?
Antibodies lacks the C- terminus transmembrane segment (hydrophobic) and therefore can be secreted and not membrane bound as BCRs are.
Describe the structure of antibodies.
Antibodies are quaternary proteins that consist of two sets of identical chains - two heavy (H) chains and two light (L) chains connected by disulfide bonds. The Fc region (the base of the heavy chain) determines the effector function (Ig class) of the antibody and is constant. The top part of the H chain, and the L chain have a constant region at the base (C terminal) and a variable region at the top (N-terminal). The variable regions make up the antigen binding site (very specific).
The variable region of both the light and heavy chain of antibodies have a lot of sequence variability, but the constant parts of both the H and L chain can also differ, how?
The light chain constant region have two isotypes, kappa and lambda. The H chain constant region have five isotypes: alfa, my, gamma, delta and epsilon. These isotypes are used to divide antibodies in different classes, as they are able to bind to different things.
The constant region of the Fc part of antibodies have five isotypes, which?
alfa, my, gamma, delta and epsilon. These isotypes are used to divide antibodies in different classes: IgA, IgM, IgG, IgD and IgE, each with different properties and effector functions.
Describe the structure and function of IgA.
IgA: monomer/dimer (most common), trimer or tetramer. The main class of antibody found in many body secretions, including tears, saliva, respiratory and intestinal mucosa. Capable of dimerization (and polymerization) which provides the possibility of lower affinity but higher avidity, can bind more different antigens with the same strength as more specific antibodies. less stable than IgG. Transported via poly Ig receptor transporter in mucosa.
Describe the structure and function of IgG.
IgG: monomer, classic Y shape with two identical heavy and light chains that is the most abundant in serum. Several (and most) subclasses with differing number and arrangement of the interchain disulfide bonds linking the heavy chains, each with different effector functions. The only antibody that is able to cross the placenta.
Describe the structure and function of IgD.
IgD: monomer, very similar structure as IgG, present on the surface of most B cells but very low abundance overall. present at higher levels in secretions in the upper respiratory tract. IgD can also induce mast cell and/or basophil degranulation (as igE) for example antimicrobial peptide release.
Describe the structure and function of IgE.
IgE: monomer, Have a similar structure to IgG but with no hinge region and have an extra constant domain that gives it the ability to bind to the surface of basophils and mast cells . When these bind antigen it induces release of among other things histamines, which make these problematic in allergy. Also have a role in combating parasitic infections (worms especially)
Describe the structure and function of IgM.
IgM: monomer when membrane bound, pentamer when secreted. No hinge region. The first class of antibodies to be produced by B-cells when maturing, they have low affinity but high avidity - strong collective binding) As they are secreted they bind together as a pentamer (with 10 binding sites) that bind pathogens in the bloodstream and clump them together in immune complexes, making it an easier target for macrophages.
Explain the structure and components of the BCR complex.
The BCR complex consist of the BCR itself (membrane bound antibody) and coreceptors and associated molecules.
- CD molecules: CD21 - involved in antigen binding (coreceptor) through binding to C3d a molecule involved in the complement system, CD19 - coreceptor to CD21 and CD81 - involved in signal transduction.
- The BCR complex include a heterodimer called the Igα,Igβ complex (CD79α,β) that contain intracellular ITAMs that transduce the signal intracellularly (since the intracellular part of the BRC itself it needs help to convey the signal)
The Igα,Igβ heterodimer in the BCR complex contain ITAMs. Brifely describe the structure of the protein and exaplain what ITAMs are.
Igα and Igβ are transmembrane proteins with short, N-terminal regions and long intracytoplasmic tails containing regions called immunoreceptor tyrosine-based activation motifs, or ITAMs. ITAMs are short sequences of amino acids that include two tyrosine residues placed approximately 10 residues apart. These tyrosine residues become phosphorylated when the associated BCR molecule is activated by binding to its ligand and can serve as docking stations for other signalling molecules which can relay the signal further.
Briefly describe the structure of TCRs.
TCRs consist of two non-identical chains (heterodimer) that are different but both contain two Ig domains, where the top one (α/β1) makes up the variable region and the lower one (α/β2) makes up the constant region, and a transmembrane domain. The variable region is the antigen binding site and have three CDRs forming peptide-specific binding site. But for TCRs to bind the antigen, it needs to be presented on MHC molecules. TCRs have the coreceptors CD4 and CD8 to bind either MHC II (CD4) or MHC I (CD8) molecules. These coreceptors also contain Ig domains and increase the avidity of antigen binding. The TCR complex binds to both the antigen and the MHC molecule it’s presented on through non covalent interactions. (and additional signals are needed for activation).
Note: The two chains of the TCR itself can be either αβ and γδ depending on cell type, but αβ is the “standard” one that binds MHC molecules and γδ binds non-MHC molecules and is more common during fetal development.
TCRs are not able to convey the signal itself because of their short cytosolic domain, what is included in the TCR complex?
Besides the CD4/8 coreceptor, the TCR complex also needs help with conveying the signal because of their short cytosolic domain, they use the CD3 complex - consisting of three pairs of Ig domain chains with ITAMs (similar to the Igα,Igβ complex in the BCR complex but bigger) to relay the signal intracellularly.
The TCR complex also include the CD28 coreceptor which binds CD80 or CD86 present on activated APCs (that have internalized antigen), a critical signal for T cell activation.
What is the basis of why the innate immune system respond fast and the adaptive immune system respond slow (7-10 days)?
B and T cells only express one receptor variant on each cell with a huge diversity between cells, which means that in order to respond to the pathogen optimally, they need to proliferate a lot - which takes time (clonal expansion). In contrast to the adaptive immune cells, the cells in the innate immune system expresses several different innate receptors on each cell, which means a lot of cells already have the receptors needed and don’t need to proliferate to respond effectively (non-clonal expression). Also, many additional cells that aren’t immune cells express innate receptors, which contribute to the fast response.
How does the innate receptors recognize pathogens?
The innate receptors are called pattern recognition receptors (PRRs) and recognize PAMPs, pathogen-associated molecular patterns. PAMPs are motifs of recurring patterns of bacteria, yeast, parasites and viruses. Innate receptors can be present both on the cell surface as the adaptive receptors, but also on intracellular membranes like endosomal membrane or even in the soluble portion of the cytosol. The makeup of innate receptors differs for different cell types.
Some innate immune receptors are also capable of recognizing antigens associated with dead or dying cells, referred to as damage-associated molecular patterns (DAMPs).
What are cytokines?
Cytokines are proteins that communicate among cells of the immune system, often called the hormones of the immune system (because as hormones, they can have endocrine, paracrine and autocrine action). Cytokines bind to cytokine receptors non-covalently but with extremely high affinity, so even just a few cytokines can mediate strong responses in the receiving cell. The response is often a change in the transcriptional program of the target cell with a wide variety of outcomes.
Chemokines are a type of cytokines, what is their function?
Chemokines act as chemoattractants, meaning cells with chemokine receptors move towards higher concentration of them. So they aid in recruitment of immune cells to infected locations and are the main drivers of local responses.
Cytokines are pleiotropic, redundant, can be synergistic or antagonistic and can lead to cascades. Explain these terms in relation to cytokines.
Pleiotropic: Cytokines can induce different biological effects depending on the target cell.
Redundant: Many different cytokines can mediate the same response in different cells.
Synergistic: The response of the combination of two cytokines can result in a bigger response than their sum.
Antagonistic: One cytokine can inhibit the effect of another.
Cascade effects: Cytokine signalling to a target cell can cause it to produce
additional cytokine(s).
There are five families of cytokines (excluding chemokines) which are they and what are their main effect?
Interleukin-1 family (IL-1): Important inflammatory mediators that are secreted very early in immune responses by macrophages and dendritic cells. They can act both locally and systemically and can help activate the adaptive response.
Class 1 (hematopoietin) cytokine family: Very diverse group. For example IL-2 that mediate interactions between leukocytes and enhance their function. IL-4 has many roles: stimulate proliferation of B and T cells and differentiation of plasma cells for example.
Class 2 (interferon) cytokine family
For example IFN-α that is important in antiviral responses, IFN-γ that is produced by activated T/NK cells and is a potent modulator of adaptive immunity and IL-10 which is anti-inflammatory.
Tumor necrosis factor family: involved in immune system development, effector functions, and homeostasis.
Interleukin-17 family: Pro-inflammatory and drive neutrophil accumulation and activation in local responses.
Everything cytokines: page 219
How can cells change their susceptibility to actions caused by ligand-receptor binding?
A cell can become more or less susceptible to actions of a ligand by
increasing or decreasing expression of the receptor for that ligand. For example cytokines can mediate this.
Remember: All different signals in the receiving cell are integrated into a network of intracellular signalling - allows for fine tuning and redundancy.
Antigen-mediated receptor clustering initiates signaling in B and T cells, which often causes receptor dimerization (or multimerization). Where are these clusters located?
Clustered receptors are localized in lipid rafts.
The estimated number of different BCRs/TCRs generated exceeds 10^13. How is this possible when our genome has aprox. 25000 genes? How come we have the capacity to tailor an antibody to any new antigen we encounter?
These were questions that puzzled scientists for a long time, and the answer lies in the BCR and TCR gene orrganization. The genes for these receptors consists of a big repertoir of fragmens that can be used to build the receptors in different combinations, and once a successful combination is made, the rest is removed, so that only that specific combination is present - providing the possibility of expanding that particular combination if needed.
Are the different combinations of lymphocyte receptors inherited?
No! Then it would not be possible for the offspring to continue making new combinations. All the different fragments are encoded for in the offspring and then recombined B/T cells.
Explain the different gene families and segment types used for light vs heavy chains of antibodies.
Light chains have two types, kappa and lambda. Kappa light chains are encoded on human chromosome 2 and contain the V (variable), J (joining) and C (constant) segments. In humans, we have
lambda light chain genes are encoded on human chromosome 22 and consist of V segments and pairs of J - C segments.
Heavy chain genes are encoded on human chromosome 14 and consist of the V, D (diversity), J and C segments.
Recombination among the various variable region gene segments generates a diverse repertoire of antibody combining sites. So, a developing B cell combines either Vκ, Jκ or Vλ, Jλ as a light chain variable region gene, and recombines one VH, one D, and one JH segment to form a heavy-chain variable region gene.
Remember that even though we talk about V, J and C segments for all, it not the same segments, they are encoded on different chromosomes and encode for different amino acids, they are just called the same for simplicity. Also, the order is from upstream do downstream, so don’t write J, V, C for example.
Which are the key enzymes involved in V(D)J combination?
V(D)J recombination is carried out by a collection of enzymes collectively called V(D)J recombinase, the key enzymes involved are recombination activating genes 1 and 2 (RAG), terminal deoxynucleotidyl transferase (TdT), and Artemis nuclease, a member of the ubiquitous non-homologous end joining (NHEJ) pathway for DNA repair.
Explain the mechanism of V(D)J recombination in short
During a recombination event, the VDJ recombination machinery is guided to recombination signal sequences (RSSs) flanking the variable (V), diversity (D), and joining (J) genes segments.
RAG1 binds an RSS, RAG2 is recrited to stabilize the RAG1/2 complex nicks the DNA in the RSS before the (V, D or J) segment. The segments are then fused to form a gene only containing one V (one D) and one J segment.
Full explaination on page 471-474.
Why is V(D)J combination so highly regulated?
Because V(D)J recombination entails the cutting of DNA at both strands, and because inappropriate recombination is potentially catastrophic for the cell, mechanisms have evolved that restrict antigen receptor gene recombination events to the appropriate sites on the Ig genes and to ensure that they occur only during defined periods of B and T cell development.
Describe the mechanisms which regulate V(D)J recombination.
- V(D)J recombination is sequential, everything happens in a specific order to ensure that nothing goes wrong. Both on chain segment recombination level but also the order in which the chains are recombined.
- The recombination signal sequences (RSSs) provides specificity, so that only the Ig genes are subject to the recombination. It also ensures that there is always one of each segment in the final genes.
What five mechanisms contribute to the diversity of antibodies in naive B cells?
- The large number of gene segments (V, D, J)
- The combinatorial possibilities of the heavy chain/light chain
- P nucleotide addition―templated nucleotide addition between joints, resulting from asymmetrical cleaving of hairpin structures
- Exonuclease trimming―sometimes occurs at junctions, losing nucleotides and changing reading frames
- Nontemplated N nucleotide addition―mediated by TdT activity, adding in random nucleotides between joints
(Then, for activated B cells, the diversity continues in germinal centres - Somatic hypermutation: Activation-induced cytidine deaminase, AID can deaminate cytidine into a uracil which is converted to a thymine, eventually leading to the change from a C-D to a T-A base pairing.)
Explain the concept of allelic exclusion in BCR expression.
Since not all combinations are successful, the cell tries with both alleles for the heavy chain if needed, and both alleles for the kappa and lambda chain is needed. Once a successful combination is done, the other allele is abandoned and the cell moves on to the next step, eg if H chain is done with allele 1, it moves on to the light chain directly and abandon/exclude the other allele. Allelic exclusion ensures that each B-cell synthesizes only one heavy and one light chain.
Explain the order in which the BCR gene is recombined during development.
The heavy chain is recombined first, starting with ligation of a D and J segment, followed by joining of the V segment to the D-J pair. After successful recombination it’s paired with a “surrogate” light chain as it is not stable on it’s own. Then the kappa light chain is recombined, if successful further recombination is inhibited, if not, the other allele is tried. if not succesful the lambda light chain is recombined and so on, until a successful heavy-light chain combination have been completed.
If no heavy chain recombination is successful the cell dies through apoptosis, same goes if no light chain recombination is successful.
What happens if a BCR turns out to be autorreactive?
A functional antibody in an immature B-cell may bind to self-antigens, but the recombination machinery can be turned back on to edit original rearrangement to salvage the rearrangement or at least inactivate it. This receptor editing of potentially autoreactive receptors only occurs in light chains.
Explain the different gene families and segment types used for light vs heavy chains of TCRs.
The T cell receptors are heterodimers consisting of one heavy and one light chain, there are two versions of TCRs, where the majority of T-cells use αβ TCR and a minority of T-cells use γδ TCR, but the two are structurally similar. Each chain consists of one variable and one constant region domain.
The light chains are α and γ variable regions encoded by V and J segments.
The heavy chains are β and δ variable regions encoded by V, D and J segments.
The recombination process is really similar to that of BCR genes.
Do TCRs undergo somatic hypermutation?
No! TCRs are not secreted and don’t undergo somatic hypermutation. Considering that cytotoxic T cells kill cells, it would be far too dangerous if a somatic mutation resulted in autoreactivity.
How does the allelic exclusion in TCRs differ from that of BCRs?
Allelic exclusion is not as complete in T cells as in B cells, in that some T cells bear more than one α chain, but the likelihood of these cells being able to recognize more than one antigen is very low given the complexity of T Cell antigen recognition.
it should be noted that the presence of even one αβ TCR capable of binding self-antigens at high affinity would result in the elimination of the cell, irrespective of the specificity of the receptor formed using an alternative α chain.