Immunology: Adaptive Immunity

CD4+ T cell activation how do they get activated?

-APC presents antigen on MHC class II -the corresponding CD4+ T cell that recognizes the MHC-peptide combination will be activated

CD8+ T cell activation how do they get activated?

-APCs present viral peptides on both MHC class I and MHC class II -naïve CD8+ T cell recognizes the MHC class I -viral peptide combination will then be activated and will proliferate

structure of T cell

-All T cells (both CD4 and CD8) have antigen receptors known as the T cell receptor (TCR) => the TCR is analogous to the antibody -variable region (= antigen binding site) -constant region

isotype class switching (class-switch recombination)

-B cells undergo isotype class-switching to make the more effective isotype for the given infection (usually IgG)

T cells will express various surface molecules at certain times of development:

-Early on in development the T cell lacks both CD4 and CD8, which is why they are referred to as double-negative -It will then express CD3, which continues to be expressed on all T cells -The T cell will then express both CD4 and CD8 on the surface (double-positive) -Once positively selected, the T cell will then commit to either CD4 or CD8 lineage (single-positive).

IgE in allergies

-IgE will develop an antigen binding site for a parasite or for a completely benign protein that someone can become allergic to => once the B cell makes IgE, it will bind to an IgE receptor and will hang out on the surface of a mast cell that has a lot of these IgG receptors -If the pathogen or allergenic protein isn't encountered, then that mast cell will sit at rest. -If the antigen IS encountered, there will be cross-linking of IgE, and that will send signal to mast cell to become activated and to send off all the granules with the mediators that will give rise to symptoms

positive selection

-T cells are selected for continued maturation -T cells need to recognize the host's MHC molecules AND pathogen peptides -so, a T cell that doesn't bind at all to a host MHC molecule would be useless, and therefore should be eliminated -only those T cells with TCR that recognizes self MHC will be positively selected.

effector T cells

-TH1 cells will secrete interferon-γ (IFN-γ) to help macrophages be more efficient at killing organisms within the macrophages. -TH2 cells secrete cytokines that promote B cell activation, and particularly the production of IgE, which have a role in immunity against parasites as well as allergic diseases. -TH17 cells promote the recruitment and activity of neutrophils -TFH (follicular helper) cells are the main helper T cells that activate B cells to become plasma cells, and to undergo affinity maturation and class switching -Treg (regulatory) cells secrete cytokines that dampen the immune response

T cell development in the different regions of the thymus:

-The double negative T cells enter the thymus through the venules -The double negative T cells then start to migrate through the thymus while rearranging their VDJ gene segments to make T cell receptors -The double negative T cells will then proliferate and become double positive T cells. => During this process, the developing T cells are in close contact with thymic cortical epithelial cells, which have the ability to display MHC with self-peptides -T cells that bind to a self-MHC molecule (with enough affinity for positive selection) will then be positively selected -If the MHC that the given T cell recognizes is an MHC class II, then the T cell will commit to the CD4+ lineage -If the MHC that a given T cell recognizes is an MHC class I molecule, it will commit to being CD8+.

major histocompatibility complex (MHC)

-aka Human leukocyte antigen (HLA) -our MHC repertoire is determined by genetics -the two main MHC molecules are MHC class I and MHC class II

the result: diversity

-at the DNA level of the light chain locus (either κ or λ), there are several V gene segments to choose from, and several J gene segments to choose from

what's the best T cell response to deal with a cytosolic pathogen (e.g., like a virus)?

-best T cell response to deal with a cytosolic pathogen is to kill off the infected host cell to prevent further viral replication and spread -CD8+ cytotoxic T cell would work best -good thing is that ALL of our nucleated T cells have the ability to present viral peptides on MHC class I to alert activated cytotoxic T cells to induce apoptosis of the infected cells **e.g., when a virus infects, it enters the cytosol of the host cells to replicate and produce more virions to spread to adjacent cells

what's the best T cell response to deal with a intravesicular pathogen (e.g., mycobacteria and fungi)?

-best T cell response to this type of infection would be to provide help to the macrophage to more effectively kill the intravesicular pathogen => T helper response, such as TH1, would be ideal (CD4 T cell) -in order for TH1 cells to achieve this, macrophages that have engulfed such pathogens can display the pathogenic peptides on MHC class II to alert the TH1 T cells to help them -IN PIC: in this example, a macrophage engulfs a pathogen and attempts to degrade it within the vesicles. However, some intravesicular pathogens, such as mycobacteria and fungi, are difficult to kill even within these vesicles

what's the best T cell response to deal with extracellular pathogens and toxins (e.g., most bacteria)?

-best response to this type of pathogen would be to generate a strong antibody response -but B cells need T cell help to do this... B cells can take in the pathogen or its toxins via receptor-mediated endocytosis, and present the peptides on MHC class II to alert TFH cells to come further activate B cells to become antibody-secreting plasma cells

T cell differentiation

-depending on the type of pathogen, the T cell clones will differentiate into one of several effector T cell types -memory T cells are also generated.

B cells

-develop in bone marrow -make antigen receptors that are expressed at the cell surface -these antigen receptors (aka immunoglobulins or antibodies) recognize a unique epitope

B cell development: where do they develop?

-develop in bone marrow -some will also develop in the fetal liver and neonatal spleen

junctional diversity

-during the process of VDJ rearrangement, some nucleotides can also be either added in or deleted at the sites where gene segments are combined together -this can further create changes in the resulting antigen-binding site

T cell receptor (TCR)

-formed by an α and β chain -a given T cell will express many TCRs that all have the same antigen binding site (analogous to the B cell that only expresses antibodies that all share the same antigen binding site)

structure of antibody

-has constant and variable region -variable region makes up the antigen-binding site **a single B cell can make many antibodies, but they all have the same antigen-binding site

negative selection

-if the double positive thymocyte's TCR binds too strongly to self- peptide:self-MHC complex, it dies by apoptosis -antigen presenting cells (dendritic cells and macrophages) are important in negative selection

IgA

-important in respiratory and gut mucosa -found in secretions and other body fluids such as tears, saliva, breast milk

cytotoxic T lymphocytes (CTLs)

-induce apoptosis, programmed cell death, of their target cells -this can be done via the Fas-FasL (Fas-ligand pathway) or through the perforin and granzyme pathway

IgE

-involved in acute allergic reactions and protection against parasites -binds mast cells and basophils

IgM

-large, pentameric -important in activation of complement -first antibody produced in the immune response -elevated IgM titres imply more recent infection

professional antigen presenting cells

-macrophages -dendritic cells -B cells **Professional antigen presenting cells can express both MHC class I (because they are also nucleated cells) and MHC class

MHC class II

-only present on professional antigen presenting cells -made up of 2 α and 2 β- chains -there are 3 pairs of MHC class II α- and β- chain genes: HLA-DR, - DP, DQ.

MHC class I

-present on all of our nucleated cells -made up of 3 α chains with a molecule called β2-microglobulin -there are 3 MHC class I α-chain genes called HLA-A, HLA-B, HLA-C.

IgD

-produced early in immune response (with IgM) -can also fix complement -present in very low numbers (generally not measured)

plasma cells

-secrete antibodies -some plasma cells will survive for only a few days to weeks, whereas others can persist for years

IgG

-smallest and most abundant -most important isotype in protection from infections => neutralization and opsonization -crosses the placenta

the fate of B cells once activated:

-some of the clones of the activated B cells will indeed become plasma cells -some of the B cell clones will become resting memory B cells. the memory B cells will continue to populate lymphoid tissue and circulate in the blood

B-cell activation by antigen and helper T cells:

-the B cell's surface antibodies can bind to a pathogen -it can then take in the antibody-bound pathogen, degrade the pathogen into smaller peptides, and present them to the cell surface via a different receptor called major histocompatibility complex (MHC) -a CD4+ helper T cell that has the specific T cell receptor that recognizes the B cell-generated pathogenic peptide on the MHC molecule will then be activated to secrete cytokines that further help the B cell become activated -this process of B cell activation requires the binding of CD40 on the B cell with CD40L (CD40 ligand) on the T cell

memory B cells

-the memory B cells will continue to populate lymphoid tissue and circulate in the blood -if the infection is not cleared with the primary antibody response, or if the antigen is encountered even years later, then the memory B cells, which have already had the chance to class-switch, can quickly be activated to secrete antibodies

timeline of T cell production

-the thymus is developed by birth, and T cell production continues at a rapid rate until puberty -after puberty, the thymus begins to shrink, and the production of new T cells declines significantly

what happens when a virus infects the host cell?

-the viral proteins are made in the cytosol, using the host cell's ribosomes

TREC (T cell receptor excision circle)

-these can be used as a marker for T cells that have recently emigrated from the thymus -TRECs can be used in newborn screening to identify babies who have not made functional T cells, which is the characteristic feature of severe combined immunodeficiency (SCID) => without a bone marrow transplant, this is a fatal immunodeficiency characterized by the lack of T cells and often other cells (B cells and NK cells) => many forms of SCID (there are several genetic defects that can give rise to this clinical syndrome) will result in absent or very low TRECs.

negative selection

-those T cells that recognize self- antigens too strongly have the potential to lead to self-destruction -as a result, T cells also undergo negative selection

bottom line about these HLA molecules...

-we all have a certain set of HLA molecules on our cells -the HLA types that we each express can have implications in matching donors and recipients for tissue transplantation

MHC class II peptide loading

-when a pathogen is taken into a professional antigen presenting cell, the resulting pathogen ends up within a vesicle (rather than in the cytosol) -acidification and activation of enzymes will then degrade the pathogen's proteins into smaller peptide fragments -once the MHC class II molecules are assembled at the ER, they will be released from the ER via vesicles that will then fuse with vesicles containing pathogen peptides -the peptides are then loaded onto the MHC class II and exported to the cell surface

MHC class I peptide loading

-when a virus infects the host cell, the viral proteins are made in the cytosol, using the host cell's ribosomes -some of those viral proteins are then cleaved into smaller peptide fragments in the proteasomes within the cytosol -the viral peptides are then transported into the ER where the forming MHC class I molecule is also located -once the peptide is loaded onto the MHC class I molecule, the MHC-peptide is then released and exported to be expressed at the cell

but antigen alone is not enough to activate a naive T cell.... There are 3 requirements for T cell activation:

1. Binding of the T cell receptor to the corresponding MHC- antigen peptide on the antigen presenting cell. 2. Binding of co-stimulatory molecules (CD27 on the T cell binding to B7.1 and B7.2 on the antigen presenting cell) 3. Cytokines released from the antigen presenting cell

QUESTION What is the first antibody isotype produced by a B cell? 1. IgM 2. IgG 3. IgA 4. IgE

1. IgM

QUESTION: What are the 3 requirements for a helper T cell to activate a B cell to generate memory or plasma cells?

1. TCR binding MHC-peptide 2. CD40L binding CD40 3. Cytokines

principles of the adaptive immune response:

1. each developing lymphocyte (B &T) generates a unique antigen receptor 2. adaptive immunity is initiated when antigens or antigen presenting cells (APC's) reach secondary lymphoid organs where lymphocytes reside 3. lymphocytes (B &T) activated by antigen give rise to clones of antigen-specific effector cells

3 main functions of antibodies:

1. neutralize toxins, 2. opsonize 3. activate complement through the classical pathway

what is the function of the adaptive immune response?

1. recognition of specific "non-self" antigens 2. generation of pathogen-specific immunologic effector pathways that eliminate specific pathogens or pathogen-infected cells 3. development of immunologic memory to quickly eliminate pathogen in subsequent infection

What is the mechanism by which vaccinations protect against infection? A. Inoculation with the antigen leads to activation of lymphocytes with differentiation into effector and memory cells B. Inoculation with the antigen leads to a robust innate immune response leading to immunologic memory myeloid cells C. Inoculation with the antigen leads to activation of mast cells that produce antibodies D. Inoculation with the antigen leads to activation of B cells that produce IgE antibodies to fight infection

A. Inoculation with the antigen leads to activation of lymphocytes with differentiation into effector and memory cells

in addition to helping Th1 cells, do you remember what other function CD40-CD40L are important for?

Bcell activation by helper T cells!

The CD4 and CD8 co-receptors determine what MHC class a given T cell will recognize:

CD4+ T cells will recognize antigen peptides that are presented by MHC class II molecules CD8+ T cells will recognize antigen peptides that are presented by MHC class I molecules **Rule of 8: 4x2=8 8x1=8

what happens during gene rearrangement in T cells?

During gene rearrangement, the excised DNA forms an excision circle, known as a TREC

overview of adaptive immune response:

FIRST ENCOUNTER: -the first time an individual encounters a given pathogen, it takes time for the adaptive response to become activated -if the innate immune response cannot eliminate the infection and the pathogenic antigen levels rise, the adaptive immune response will be induced -B cells make antibodies, which are the very important in fighting most infections -T cells can differentiate into various effector T cells SECOND ENCOUNTER: -Once the adaptive immune response is activated for a given infection, in addition to eliminating the existing infection, the activated B cells and T cells can also develop memory (= if the same pathogen is encountered in the future, the adaptive response kicks in sooner and more effectively than the first time) -immunological memory allows us to fight off the same pathogen quickly if encountered in later years

what cytokine is required for T cell proliferation?

IL-2 **an activated T cell will produce IL-2 and will also express a high-affinity IL-2 receptor **when IL-2 binds its receptor, the T cell will go into cell cycle to rapidly proliferate via an autocrine mechanism.

Antibody can bind directly to the surface of a pathogen or can directly bind soluble pathogenic antigens, such as toxins.... is this the same for T cells?

NO T cell receptors (TCRs) can only recognize its antigen if it is presented on the surface of our own host cells via a cell surface molecule known as major histocompatibility complex (MHC) **so, a given TCR has to recognize BOTH the pathogenic peptide AND the MHC molecule

If a naïve B cell (waiting in lymphoid tissue) encounters its antigen in the lymphoid tissue, the antigen will bind to the surface antibodies on the B cell. is antigen binding to the surface antibodies enough to activate the B cell to proliferate, secrete antibodies, and undergo affinity maturation and isotype class switching?

NO, the B cell needs help from a T helper cell and cytokines.

antibodies are also involved in antibody-dependent cell mediated cytotoxicity (ADCC).... what is this?

STEPS: 1. antibody binds antigens on the surface of target cells 2. antibody receptors (Fc receptors) on NK cells recognize bound antibody 3. cross-linking of Fc receptors signals the NK cell to kill the target cell 4. target cell dies by apoptosis

QUESTION Which of the following processes can occur before B cell activation by antigen? 1. Somatic recombination (VDJ rearrangement) 2. Isotype class switching 3. Affinity maturation 4. Soluble antibody secretion

Somatic recombination (VDJ rearrangement)

where do T cells arise and develop?

T cell progenitors arise in the bone marrow, but then migrate to the thymus to mature

what happens if the T cell binds too strongly with an MHC- self peptide combination?

T cell will be eliminated (negative selection) **thymic cortical epithelial cells, dendritic cells and macrophages play a role in negative selection

linked recognition

The rule that for a helper T cell to be able to activate a B cell, the epitopes recognized by the B cell and the helper T cell have to be derived from the same antigen

let's look at how the antigen binding sites are encoded... (remember that each light chain and heavy chain is made up of both variable and constant regions) what segments are the variable regions further divided into?

V and J segments for the light chains V, D, J segments for heavy chains

T cell receptor diversity

VDJ somatic recombination **T cells undergo rearrangement for their α- and β chains -The α- chain is analogous to the light chain of the antibody, in that it is made up of V and J gene segments -The β- chain is analogous to the heavy chain of the antibody, in that it is made up of V, D, and J segments

in the secondary antibody response, will the memory B cells still need T cell help?

YES -however, the memory B cells have properties that allow for more efficient activation

how to make a variable region for the light chain:

a single V segment must join with a single J segment to then be transcribed and translated

epitope

a site on an antigen recognized by an antibody or a T cell receptor

antibody/B cell antigen receptors/immunoglobulins

a substance produced by the body that destroys or inactivates an antigen that has entered the body

CD4+ T helper cells

activate B-cells and release cytokines that stimulate other immune cells

Both affinity maturation and isotype class-switching are made possible by various enzymes that break DNA, add in or remove nucleotides, and repair the DNA to give rise to the new exon One of the necessary enzymes that initiates DNA breaks in these processes is:

activation-induced cytidine deaminase (AID) **Individuals who lack AID are unable to undergo affinity maturation and isotype class switching.

what does the ability of these specialized cells to present on both MHC classes allow for?

allows for some CD4+ T cells to also be activated to boost the co-stimulatory activity needed for CD8+ T cell activation

so how do CD4+ helper T cells and B cells get activated by the same pathogen?

bc the B cell can also act as a professional antigen presenting cell

how is the ultimate effector function of the activated CD4+ T cell determined?

by additional specific cytokines that are secreted by the antigen presenting cells e.g., IL-6 promotes TFH cells **the effector CD4+ T cells will then go on and carry out their unique functions (shown in pic)

although B cells cannot phagocytose in the way that macrophages and dendritic cells can, how are they are able to engulf pathogens?

by receptor-mediated endocytosis

QUESTION: A 32 year‐old refugee from North Korea presents with cough, fevers, and night sweats. His chest x‐ ray shows bilateral opacities. His sputum acid fast bacilli smear is positive for Mycobacteria. You recall that Mycobacteria have virulence mechanisms that allow them to persist within phagolysosomes upon phagocytosis. Which of the following T cell responses would be most important in fighting off this infection? a) Antigen presentation on MHC class I to promote a cytotoxic T cell response b) Antigen presentation on MHC class I to promote a TH2 response c) Antigen presentation on MHC class II to promote a TH1 response d) Antigen presentation on MHC class II to promote a cytotoxic T cell response

c) Antigen presentation on MHC class II to promote a TH1 response

QUESTION: Which of the following best describes the purpose of VDJ rearrangement (somatic recombination)? o a) Determines the isotype of the antibody o b) Determines the constant region of the antibody o c) Determines the antigen binding site of the antibody o d) Determines the effector function of the antibody

c) Determines the antigen binding site of the antibody

isotope

constant region of the antibody - determines the effector function of the antibody

when are B cells produced?

continuously produced, even in adulthood

what will most CD8+ T cells become?

cytotoxic T cells, which kill virus-infected host cells

CD8+ T cell activation once activated, what do most CD8+ T cells differentiate into?

cytotoxic T lymphocytes (CTLs) that can kill viral-infected host cells

when does somatic recombination occur?

during B cell development **when a B cell is first developing, it will randomly create its antigen-binding site through somatic recombination

when does T cell undergo somatic recombination?

during T cell development (in the thymus)

there are several proteins necessary for somatic recombination to occur:

e.g., RAG-1 and RAG-2

how to make a variable region for the heavy chain:

for the heavy chain, a single V segment will join with a single D segment and a single J segment

Th1 cells

help macrophages kill engulfed pathogens that persist in the macrophage vesicles **binding of CD40L on the T cell to the CD40 on the macrophage as well as secretion of IFN-γ are critical for this function

affinity maturation

if the B cell is activated (by encountering its antigen in an infection, with the help of T cells, and with help from cytokines), it can make some minor edits to its antigen binding site in an attempt to make the antibody even more effective ***point mutations are introduced into the rearranged V-region genes to select for more effective antibodies with improved affinity for antigen

what does a B cell need to do in order to survive?

in order to survive and make it through the various stages of development, a B cell needs to make a functional antibody

using immunological memory for vaccines...

inoculation with a pathogen (or pathogen component) to develop long-term immunity -of course, there are many exceptions, as pathogens can also develop sneaky ways to evade the immune response

once the B cell is activated, what can it do?

it can proliferate into clones that are capable of secreting antibodies, undergoing affinity maturation, and class switch recombination.

diversity in antibody repertoire:

it is because of the countless potential combinations that these V-J (light chains), and V-D-J (heavy chains) segments can give rise to, that allows for so much diversity in antibody repertoire

what happens once a B cell is activated?

it will proliferate and differentiate into plasma cells (which can then secrete the immunoglobulin in soluble form)

what happens once the T cell is activated?

it will proliferate to give rise to many T cell clones that have the same T cell receptor

what is required for a B cell to become activated?

its cell surface immunoglobulin must bind to antigen, it must have the help of T helper cells, and it must have the necessary cytokines

Some of the isotypes are better at certain functions that others... what are some of the main ones to look out for?

o IgG -most important in clearing infections -very good at neutralizing toxins and viruses, opsonizing, activating complement, and initiating ADCC -can cross the placenta (from mother to fetus). o IgM -excellent at activating complement o IgE -binds to mast cells and is important in the response against parasites -activates and degranulates mast cells (key players in many allergic diseases) o IgA -typically found in secretions (breast milk and tears) and lumen (e.g., gastrointestinal tract) as it can be transported across the epithelium

at what point can CD4+ or CD8+ naïve T cells become effector T cells?

only once they encounters its antigen

T cells

produced in bone marrow, mature in thymus => T cells further differentiate into different effector T cells, based on the expression of either CD4 or CD8 co-receptors

QUESTION: Point mutations can be introduced into rearranged V-region genes to select for more effective antibodies with improved affinity for antigen. What is this process called?

somatic hypermutation or affinity maturation

-Remember that a single antibody can only recognize a single epitope -You also learned that a single B cell can only make antibodies that all have the same antigen-binding site -Think of the billions of epitopes for all of the potential pathogens that our antibodies have to be able to recognize! How is this diverse repertoire of antibodies generated?

somatic recombination => gene rearrangement that occurs in B and T cells

HUMORAL IMMUNITY

specific immunity produced by B cells that produce antibodies that circulate in body fluids

what happens if the immature B cell, with its surface IgM, binds very strongly to a self-antigen while it is still in the bone marrow?

the B cell will not survive

both positive and negative selection of T cells:

the T cells that make it through both positive and negative selection will then go on to mature and migrate to other lymphoid tissue

what happens if the innate response is not enough?

the adaptive immune response, which is carried out by B cells and T cells, provides another layer of defense against infection -the adaptive immune response requires a coordinated sequence of events in order to become activated

how do we produce B cells that make antibodies that only recognize pathogenic antigens? how do we prevent the production of antibodies that would bind to our own self-antigens and attack our own tissue?

the immature B cells are tested for autoreactivity before they are released into circulation **do this via negative selection

how does the host cell know which type of MHC molecule to present the pathogenic peptide on?

the way a peptide is loaded onto an MHC class I molecule is quite distinct from how a peptide is loaded onto an MHC class II molecule... so they're distinguishable

what happens once we make a variable region for the heavy chain?

then a rearranged light chain will join with a rearranged heavy chain to give rise to the antibody with its specific antigen-binding site

antibody isotypes

there are 5 different antibody isotypes: IgM, IgD, IgG, IgE, IgA

what happens to B cells that do not bind strongly to self-antigens in the bone marrow?

they migrate to peripheral lymphoid organs -they must then enter follicles within lymphoid tissue in order to survive -the B cells within lymphoid tissue are then ready to be activated, with the help of T cells, if their antigens are encountered

what happens to all the intervening DNA in between the joined V and J segments?

they will be excised out (hence, the gene segments are rearranged)

what will most CD4+ T cells become?

they will differentiate into a variety of effector T cells

exception to the rule.... some potent antigens are capable of activating B cells without the help of T cells. what are these antigens called?

thymus-independent antigens e.g., bacterial capsular polysaccharide.

goal of somatic recombination?

to generate a TCR with a specific variable region that determines the antigen binding site

CELL-MEDIATED IMMUNITY

type of immunity produced by T cells that attack infected or abnormal body cells

T cells that make it through positive and negative selection:

will continue on as either CD4+ or CD8+ naïve T cells (naïve because they have not yet been activated)

antibody structure: review

• 1 heavy chain -includes constant region, which determines the isotype of the antibody -each heavy chain is also made up of constant and variable regions • 2 light chains -either κ or λ -each light chain is made up of constant and variable regions => the variable region (= antigen binding site) is determined by both heavy and light chains (C = constant, V = variable, L = light, H = heavy)

perforin and granzyme

• perforin = a molecule that forms pores in the membrane of the target cell • granzymes = enzymes that can induce apoptosis -can then enter the pore formed by perforin to kill the target cell.