Effector responses

Question 1

There are two types of effector responses, which?

Answer 1

The humoral effector responses: antibodies, clears extracellular pathogens.

The cell mediated effector responses:
- antigen specific cytotoxic T cells (CTLs) and NKT cells

• antigen non-specific NK cells, clears intracellular pathogens. (Some populations of CD4 T cells can be cytotoxic, producing cytokines that cause cell death.)
• Cells such as macrophages, neutrophils, eosinophils, and NK cells all express Fc receptors, which can induce
  phagocytosis of antibody-antigen complexes and/or the direct killing of target cells via a process known as antibody-dependent cell-mediated cytotoxicity.

These responses are different but still have overlapping roles in clearance of the infection in the host.

Question 2

What determines how an antibody clears the infection?

Answer 2

How an antibody contributes to clearing infection depends on its class (its heavy chain isotype), which controls some of its effector functions, including whether it can recruit complement.

The class of an antibody also determines which receptors can bind it. Antibody-binding receptors, which bind the constant regions of antibodies and are therefore
called Fc receptors or FcRs, determine which cells an antibody can activate to aid in its protective mission, as well as whether it can gain access to certain locations in the body.

Question 3

Antibodies protects the host through six antibody-mediated effector functions, which?

Answer 3

There are six antibody-mediated effector functions:
- Neutralization
- Agglutination
- Opsonization
- Complement activation
- Antigen dependent cell mediated cytotoxicity (ADCC)
- Degranulation

Question 4

Describe the antibody-mediated effector function neutralization in short.

Answer 4

Neutralization: binding to pathogens or toxins and preventing them from binding to cells and leading to infection or damage.

Question 5

What is agglutination?

Answer 5

Agglutination: cross-linking particulate antigens such as bacteria, blocking them from binding to cells or tissues and enhancing their clearance.

Question 6

What is opsonization?

Answer 6

Opsonization: binding to Fc receptors on the surface of phagocytic cells and inducing phagocytosis and killing.

Question 7

What is complement activation?

Answer 7

Complement activation: antibodies binding to pathogen and initiating the complement cascade, resulting in the generation of complement proteins that can enhance phagocytosis via complement receptors or can directly kill pathogens via the membrane attack complex (MAC).

Question 8

What is antigen dependent cell mediated cytotoxicity (ADCC)?

Answer 8

Antigen dependent cell mediated cytotoxicity (ADCC): cell-bound antibody binding to Fc receptors on cytotoxic cells (e.g., NK cells; also macrophages, neutrophils, eosinophils), activating secretion of cytotoxic proteins that induce apoptosis of or kill infected cells, tumor cells, and/or pathogens.

Question 9

What is degranulation?

Answer 9

Degranulation: antibody-antigen complexes binding to Fc receptors triggers fusion of prepackaged secretory granules with the plasma membrane, allowing release of mediators that have protective effects, such as damaging helminth parasites.

Question 10

Which antibody class(es) are capable of performing neutralization?

Answer 10

All classes: IgA, IgE, IgG, IgM and IgD are capable of neutralization. Neutralization is the most basic effector function but also offers the most effective protection against damaging effects. However, a lot of pathogens have developed evasion strategies to fight this, like invading cells or adhering to epithelial cells (away from blood where the antibodies circulate) but it’s a good start.

Question 11

Which antibody class(es) are capable of performing agglutination?

Answer 11

IgA, IgM and IgG are involved in agglutination. IgA and IgM are polymeric when secreted (more antigen-binding sites per molecule) and thus better at forming larger immune complexes that can be cleared and it’s even harder for a clump of pathogens to bind to and infect cells or tissues than it is for a single pathogen.

Question 12

Which antibody class(es) are capable of performing opsonization?

Answer 12

Mainly IgG, but also IgA and IgM are involved in opsonization. IgE and IgD are present in such low concentrations that it wouldn’t really help that much. Phagocytes don’t express Fc receptors that bind IgE or IgD.

Question 13

Which antibody class(es) are capable of complement activation?

Answer 13

IgM and some IgG subclasses have the capacity of stimulating complement activation (classical pathway).

Note: Activation of the early part of the complement cascade can also result in binding of complement components that protect the host by opsonizing pathogens.

Question 14

Which antibody class(es) are capable of performing antigen dependent cell mediated cytotoxicity (ADCC)?

Answer 14

Subclasses of IgG are mainly driving ADCC, but other antibody classes can also mediate it. The human IgG subclass IgG1 is the most potent in driving ADCC.

NK cells express the receptor for IgG Fc (specifically, FcγRIII)

Question 15

Which antibody class(es) are capable of triggering degranulation?

Answer 15

Degranulation is mostly described in the contect of IgE antibodies, but other classes are also capable of triggering it (like IgD).

Question 16

Describe the effector functions of IgM.

Answer 16

IgM is the first antibody to be secreted during an immune response, it has low affinity but in it’s secreted form it is a pentamer, with 10 antigen binding sites in total, leading to high avidity and good opportunity for immune complex formation that can be engulfed by phogocytes. It provides quick protection that can stall the infection until high affinity IgG antibodies can be produced. IgM is very good at complement fixation and opsonization.

Question 17

Describe the effector functions of IgG.

Answer 17

IgG is the main antibody to be secreted by T cell activated B cells and is synonymous with a solid immune response. IgG1 and 3 are effective in complement activation and and ADCC. All variants of IgG bind Fc receptors on macrophages that can phagocytose the ag-IgG complexes. IgG is the only antibody that is capable of crossing the placenta and thus also helps protect fetuses. It is also the most widely used therapetutically.

Question 18

Describe the effector functions of IgA.

Answer 18

IgA is found in all mucosal tissues and secretions and is very effective in neutralization and immune complex formation (agglutiation). IgA does NOT activate complement or drive inflammation, mainly because it’s present in mucus where commensal bacteria reside, that we don’t want to attack. IgA is very long-lived in mucosa due to protease-resistant amino acid sequence in Fc region and is highly glycosylated, enabling them to associate with the mucous layer which facilitates their clearance, overall fery fit for the mucosal ennvironment.

Question 19

Describe the effector functions of IgE.

Answer 19

IgE is mainly key in the protection against parasitic worms and protozoa. It is present in serum in very small quantity (0.0003 mg/ml compared to the others which are present at ~9-0,5 mg/ml) but very potent and long lived effects. It plays a key role in allergy and asthma, where it initiated degranulation of granulocytes inappropriately, leading to inflammatory responses against benign compounds and can lead to chronic inflammation like asthma.

Question 20

Describe the effector functions of IgD.

Answer 20

IgD is the least characterized and present at small quanteties (0,03 mg/ml) in serum but is moore common in the upper respiratory tract. It may activate mast cells/basophils to release antimicrobial peptides.

Question 21

Considering the different effector functions, which antibody classes would be favored in response to an extracellular vs intracellular pathogen?

Answer 21

If the pathogen is extracellular, the class switching would probably be biased towards IgG subclasses that can fix complement and opsonize pathogen in addition to enhancing innate immune cells inflammatory activity and inducing mediator release from granulocytes, or IgE if its a parasite.

If the pathogen is intracellular, class switching would be biased towards antibodies that can activate cytotoxic cells, including NK cells, which release their granule contents to kill an infected cell (ADCC) eg IgG1.

Question 22

Even thouogh antibodies themselves have direct effector functions, another mechanism is key in mediating the effector functions of antibodies, which?

Answer 22

Fc-receptors mediate many effector functions of antibodies. Each receptor is specific for the Fc of one or several heavy-chain classes and allow nonspecific immune cells to
take advantage of antibody-specificity.

Question 23

Describe how FcR signalling works in short.

Answer 23

A single antibody will not activate an Fc receptor; instead, multiple FcRs need to be cross-linked or oligomerized by binding to the multiple antibodies coating a single pathogen. This cross-linking results in the generation of either a positive or negative signal (depending on if the receptor is associated with ITAMs (activating) or ITIMs (inhibiting)) that enhances or suppresses the effector function of that cell, depending on the cell type.

Note: cells can co-express both inhibitory and activating FcRs and tune their response according to their integration of positive and negative signals. Which FcRs a cell expresses sometimes depends on the signals that the cell receives during an immune response.

Question 24

Provide three examples of FcRs and what their function is.

Answer 24

• FcγRs: bind the Fc region of IgG antibodies and are the main mediators of Ab functions in the body.
• FcαR (CD89): bind the Fc region of IgA. Contributes to pathogen destruction by triggering ADCC , phagocytosis and cell activation.
• FcεR: bind the Fc region of IgE antibodies and contrubutes to inflammation and destruction of parasites through triggering degranulation of granulocytes.
• pIgR; Polymeric immunoglobulin receptor: expressed on epithelial cells and initiates the transcytosis (endocytosis and transport) of IgA and IgM from the blood/lamina propria to the lumen of the mucosal tissues.
• FcRn; Neonatal Fc receptor: Expressed on many different cell types early in an organism’s lifespan, mainly epithelial cells. The FcRn is key to transporting IgG from milk in the intestine to the blodstream of babies which have a less mature immune system. It also transports IgG across the placenta and recycle IgG taken up by pinocytosis back into circulation in adult life.

Question 25

Describe the Fcγ receptors, which cells express them and what function is induced when binding their ligand?

Answer 25

The FcγRs bind IgG and are the most diverse group of FcRs; four families total, where three are activating (associated with ITAMs) and one inhibiting (associated with ITIMs. They are expressed by a wide range of cells but generally they: – Will induce phagocytosis if expressed by macrophages – Will induce degranulation if expressed by cytotoxic cells

Question 26

The main inhibitory FcR is FcγRIIB, describe its function.

Answer 26

FcγRIIB binds several different IgG subclasses and is expressed by many cell types, including B cells. When bound by antibody-antigen complexes, FcγRIIB sends negative signals through the BCR that inhibit further B-cell activation, alerting the B cell that sufficient antibodies are present and no further antibody production is necessary.

Question 27

Describe the Fcα receptors, which cells express them and what function is induced when binding their ligand?

Answer 27

FcαR are expressed by myeloid cells: Monocytes/macrophages, Granulocytes, & Dendritic cells - Contributes to pathogen destruction by triggering ADCC and phagocytosis - Stimulates myeloid cells to release inflammatory cytokines and generate superoxide free radicals to help kill internalized pathogens

Question 28

Describe the Fcε receptors, which cells express them and what function is induced when binding their ligand?

Answer 28

FcεRs are expressed by granulocytes: Mast cells/basophils, & Eosinophils There are two types; High-affinity FcεRI and Low-affinity FcεRII. FcεR signalling triggers a signaling cascade that releases histamines, proteases, and other inflammatory mediators. Most often associated with allergy symptoms

Question 29

Can antibodies be rejected when transplanted?

Answer 29

Not really, which is key in the transfer of IgG from mother to infant to provide protection in the early years when the immune system is still developing. It also have huge therapeutic potential because they're so similar between people and can be used to threat a plethora of diseases like psoriasis (antibodies toweards IL-17), arthritis (TNF-α receptor), and many cancers. Human or humanized antibodies can today be produced in animals to lover immunogenecity (bc antibodies differ between species).

Question 30

Which three cell types are included as cytotoxic effector cells? What is the machanism of killing for each?

Answer 30

The three types of cytotoxic effector cells are: - CD8+ cytotoxic T lymphocytes (CTLs) - NKT cells - NK cells CTLs and NK cells kill cells via cytotoxic granule release (perforins and granzymes) and FasL-Fas interactions, while NKT cells mainly use FasL-Fas interactions.

Question 31

In order to generate mature CTLs from naive Tc cells, they need to be activated. What is different for the activation of naive Tc cells vs Th cells?

Answer 31

Naive Th cells express CD4 which recognizes peptides presented oon MHC class II molecules (from extracellular sources), naive Tc cells insted express CD8 which recognizes peptides on MHC class I molecules (from intracellular sources). In order for an APC to present the peptide aquired from an extracellular source on an MHC I molecule they need to cross-present it (switch between the regular way of externally sourced antigen presentation on class II MHC, to the intracellular antigen presentation on MHC I). In order to cross present the antigen, they need to be "licenced" by Th cells. Otherwise the activation of Tc cells is the same as for Th cells, requiring the three signals: TCR engagment, costimulatory receptor engagement (CD28 on T cell with CD80/86 on APC) and cytokine signalling.

Question 32

There are two way of licencing APCs for cross presentation, which? explain.

Answer 32

The two models for cross presentation lincencing are: - Sequential: Th cell binding to the MHC II on the APC (often DC), getting activated, and licencing the dendritic cell for cross presentation (exact mechanism unknown) --> the licenced DC can then go on to cross present the antigen on a MHC I molecule and activate the naive Tc cell to differentiate into a CTL (and memory cells). - Simultaneous: That the Th cell - DC - Tc interaction happens at the same time, the Th gets activated and licence the DC for cross presentation, then immediately presents the antigen on MHC I to activate the Tc cell. This is more likely according to Eva but they arre not mutually exclusive.

Question 33

What is the polarizing cytokine for differentiation into a CTL?

Answer 33

IL-2 is the polarizing cytokine for CTL differentiation. Naive CD8+ T cells don't express much of the IL-2 receptor nor produce IL-2. Signals 1 and 2 induce expression of both the IL-2Rα chain (generating the high-affinity IL-2 receptor) and IL-2 itself (the principal cytokine required for full proliferation and differentiation of effector CTLs, including expression of granzyme B and perforin).

Question 34

Why is the activation of CTLs so stringently regulated?

Answer 34

CTLs are extremely potent cell killers, so it would be really bad if they could be activated inapproprietly. The fact that naive Tc cells don't express the IL-2R or produce IL-2 ensures that only properly activated CTLs get active, also, the need for Th cells to be activated by the antigen before and then licencing the DC in order to allow antigen recognition by the naive Tc cells to become activated is another layer of security.

Question 35

Briefly describe a method used for tracking CD8+ CTLs.

Answer 35

By using a fluorescently labeled streptavidin that associate with biotinylated peptide-MHC class I complex in a tetramer, and insert it into the model organism, we can see a fluorescent signal when CD8+ CTLs bind to the peptide-MHC I complex. This method allowed us to see that CTLs really migrate great distances into different body tissues to combat the infected cells.

Question 36

Explain the four stages of CTL mediated killing of target cells.

Answer 36

Regardless of which method is employed for the killing, there are four stages to the process: 1. The target cell presents antigen on MHC I molecule, which is recognized by the TCR-CD3 and CD8 on the CTL and starts to come closer to ultimately bind to the target cell to form a immunological synapse: conjugate formation (very intimate - sometimes referred to as "the kiss of death"). 2. The completed conjugate formation induces polarization of granules to the contact side by cytoplasmic rearrangement. 3. CTL granule exocytosis; the contents are delivered to the target cell and induces apoptosis. 4. The CTL disassociate and is recycled and can move on to kill the next infected cell.

Question 37

A solid interaction, conjugate formation, between the target cell and the CTL is crucial for CTL mediated killing. How is this strong connection upheld?

Answer 37

The conjugate formation is dependent of adhesion molecules, that form a ring around the TCRs, formed primarily by interactions between the integrin receptor LFA-1 on the CTL membrane and the intracellular adhesion molecules (ICAMs) on the target cell membrane. The TCR signals induces a conformational change in the LFA-1 molecule that is folded and low affinity before TCR signalling, to a high affinity receptor for ICAMs. This conformation change is only upheld for 5-10 minutes before it returns, also to ensure that these immunological synapses can't form without TCR recognition, on uninfected cells. It also facilitates disassociation from the target cell after the cytotoxic agents have been delivered.

Question 38

There are two pathways for CTL/NK cell mediated killing of target cells, which?

Answer 38

CTLs and NK cells can mediate killing of target cells by: - Granzyme/perforin-mediated cytolysis - Fas (CD95)/FasL-mediated lysis

Question 39

Explain the pathway of granzyme/perforin mediated cytolysis.

Answer 39

Perforin and granzyme B is delivered via exocytosis of granules, and the perforin forms pores in the target cell membrane by going through a conformational change when in contant with the target membrane, exposing an amphiphatic domain and inserting into the membrane. Granzyme B can then enter into the cell through the pores (and through endocytosis). Once in the cytoplasm, granzyme B and other granzymes initiate a cascade of reactions that activate an apoptotic pathway within the target cell, resulting in the fragmentation of target cell DNA into oligomers of 200 base pairs, which is a typical sign of apoptosis.

Question 40

Explain the pathway of Fas/FasL-mediated lysis.

Answer 40

The Fas/FasL-mediated lysis is based on surface recognition of the Fas (CD95) ligand, a transmembrane protein expressed by many cell types that belong to the TNF (tumor necrosis factor) family that can deliver a death signal when cross linked by it's ligand FasL. FasL is synthezised by activated CTLs and localize in the granule membrane and fuses to the CTL membrane upon granule exocytosis. The ligand interacts with Fas on the target membrane and triggers apoptosis through triggering cleavage of caspases which take on proteolytic activity that systematically disassemble the cell—the hallmark of apoptosis.

Question 41

Describe the experiment that was done to prove that there were two pathways for CTL mediated killing of target cells.

Answer 41

To show that there were two pathways of CTL mediated killing, they generated CTLs in two types of mutant mice, perforin KO mice and the Fas-deficient lpr strain, which cells do not express Fas. First they went to generate CTLs from a normal mouse H2b, and then took a normal mouse H2k, and killed off the lymphocytes with mitomycin C. Then they co cultured cells from the H2k mouse with CTLs generated from the H2b mouse, and the CTLs killed the H2k cells. They did the same procedure but generated CTLs in a perforin knockout H2b mouse, and looked at if these CTLs could still kill H2k cells, which they could. This indicated that there was another pathway of killing than the perforing/granzyme pathway. To see whether the other pathway was the Fas/FasL pathway, they generated CTLs from a perforin KO mouse and co cultured it with cells from the fas-deficient H2k lpr strain (but with CTLs killed), and found that the cells survived. So the perforing KO H2B CTLs were not able to kill cells that did not express Fas. This showed that there was only two pathways for CTL mediated killing of "non-self" cells. If a third way existed, the cells would not survive.

Question 42

How much of the circulating lymphocyte population is made up of NK cells? What is so special about them?

Answer 42

5-10% of the circulating lymphocytes are NK cells. NK cells don't express antigen-specific receptors (i.e., membrane antibodies, T-cell receptors) but still play a major role in immune defenses against infected cells, stressed cells, and tumor cells.

Question 43

How do we differentiate NK cells from other lymphocytes?

Answer 43

Human NK cells express CD56 (adhesion molecule) on their surface. CD56 varies in expression depending on the maturation and activity state of an NK cell (CD56 high expressers tend to produce cytokines and may differentiate into CD56 low expressers, which exhibit more cytolytic activity).

Question 44

What is the defining trait of NK cells?

Answer 44

The defining trait of NK cells is expression of a set of activating and inhibiting NK receptors: – These receptors are used to determine whether to kill a target or not, based on the balance between activating and inhibiting signals. NK cells recognize and destroy pathogen-infected cells and abnormal tumor cells. They also proliferate earlier in infection than CTLs, providing a crucial first line defense against intracellular pathogens. They also help to regulate innate/adaptive immunity by cytokine secretion.

Question 45

What induces NK cell cytotoxic activity? when?

Answer 45

NK cell cytotoxic activity is stimulated by the innate immune cytokines IFN-α, IFN-β, and IL-12, which all rise rapidly during the early stages of a viral infection. The wave of NK cell activity peaks subsequent to this rise, about 3 days after infection, long before the 7 days it takes CTL-Ps to develop into functional CTLs. So NK cells are crucial in keeping the virus infection in check before adaptive immunity kicks in.

Question 46

Explain how NK cells recognize their targets.

Answer 46

NK cells recognize their target by a mix of “missing self” and self signals: - for the “missing self” an example is the absence of MHC I molecules on the surface (strategy to evade the adaptive immune system) or MHC I molecules not presenting self peptides but something else. - For the self they have receptors for MHC I. The balance of these two signals (inhibitory and activating) determines if the scanned cell becomes a target or not. (inhibitory signals trumps the activating ones, good for tolerance). So, the NK cells express two different categories of receptors: one that delivers signals that inhibit the cytotoxic activity of NK cells (receptors that recognize MHC class I proteins) and another that delivers signals that stimulate cytotoxic activity (receptors that recognize ligands upregulated on infected, stressed, and tumor cells). NK cells distinguish healthy cells from infected or cancerous ones by monitoring and integrating both sets of signals; whether or not a target is killed depends on the balance between activating and inhibitory ligands that it expresses. NKRs don’t go through allelic exclusion - important as a person can have many different MHC molecules (polymorphisms) and thus the NK cells don’t attack healthy cells.

Question 47

How does our current understanding explain how NK cell activity is "activated" and how tolerance is maintained?

Answer 47

NK cells don’t automatically possess killing potential – It seems they’re “licensed to kill” by a prior interaction with a healthy cell through MHC class I/inhibitory receptor interactions –> This gives the “license” only to those NK cells that can exhibit restraint when encountering a healthy, normal cell. Still an area of active research for further clarification!

Question 48

Briefly describe the experiment that showed that NK cells are also capable of forming some sort of memory.

Answer 48

The researchers allowed NK cells to recognize MCMV infected cells, proliferate and expand and kill the virus infected cells and then after contraction of the immune response, they extracted the NK cells remaining and performed both in vivo and in vitro experiments testing the killing capabilities: - The MCMV primed NK cells were transferred to immunodeficient mice, in which they could protect from MCMV infection. - in vitro studies showed that these MCMV primed NK cells were more effective in killing MCMV infected cells than naive NK cells. So there is obviously some sort of memory machinery, but we don't understand it fully yet.

Question 49

The several studies that have found some sort of memory for NK cells raises exciting ideas, like what?

Answer 49

If NK cells have memory, this raises the exciting idea that it may be possible to immunize people to enhance their NK-cell memory against viruses and possibly even against certain tumors, potentially providing an expanded army of NK cells for early innate protection against infection or malignancy.

Question 50

Compare NKT cells with T cells and NK cells.

Answer 50

- NKT cells bridge innate/adaptive immune systems: Not NK cells, not quite T cells - Possess a TCR like T cells, but it is invariant, and recognizes glycolipids presented by nonpolymorphic CD1d - Can act as helper cells (secreting cytokines) or killer cells - Killing seems dependent on Fas-FasL interactions only (not perforin/granzyme too like NK/T cells) - Include both CD4+ and CD4– cell types - No memory - Possess NK surface proteins rather than T-cell varieties