Humoral Immune Responses Flashcards
LO’s #1-6 humoral immune response
- List and describe the function of receptors and their ligands on surface of B cells. Include the following:
a. BCR
b. Co-BCR
c. HLA Class II
d. CD40
e. CD80/86 (B7)
f. CD20
g. ICAM-1 - Diagram the migration of mature, naïve B lymphocytes in the lymphoid tissues.
a. Describe the role of, leukocyte function–associated antigen 3 (LFA‐3) and ICAM‐1 (intercellular adhesion molecule - Diagram the antigen‐induced first signal in B cells. Describe how each ligand interaction contributes to antigen recognition (first signal) leading to activation.
- Describe the functional outcomes of B cell activation by antigen (first signal).
a. Primary immune response
b. IgM secretion
c. Change in chemokine receptors
d. Migration in lymph node - Diagram the immune synapse between a B cell and a T cell. Describe how antigen recognition by T cells leads to second activation signal for B cell.
- Describe the changes that occur in a fully activated B cell in the extra-follicular and germinal centers (GC) of lymphoid tissues.
a. Migration in lymph node
b. Change in chemokine receptors
c. Activation induced deaminase (AID)
i. Heavy‐chain isotype (class) switching directed by cytokines produced by follicular helper T cells
ii. Affinity maturation
d. Generation of plasma cells and memory B cells
LOs #7-12 humoral immune responses
- Predict how T-independent antigens would affect B cell responses.
- Describe how properties of antibodies determine their effector function. Include:
a. Structure of Ab molecule; Fc and Fab
b. Role of isotype (class) switching and affinity maturation
c. Role of IgG isotype in prolonged ½ life of molecule - Describe the major mechanisms by which humoral immunity provides protection to the host. Include cell types, distribution of Fc receptors and class of antibody(ies) for each mechanism.
a. neutralization of microbes and microbial toxins
b. opsonization and phagocytosis
c. antibody‐dependent cellular cytotoxicity (ADCC)
d. Classical pathway of complement activation
e. IgE‐ and eosinophil/mast cell‐mediated reactions - Describe the function of IgA antibodies at mucosal surfaces.
- Describe role of antibodies in neonatal immunity.
- Describe the mechanism of contraction of a humoral immune response to homeostasis.
a. antibody feedback
reference text for humoral immune response
- Abbas: Basic Immunology 6th ed.
- Ch. 7 and 8: Humoral Immune Responses and Effector Mechanisms of Humoral Immune Responses
Additional references
- Abbas, Cellular and Molecular Immunology 9th ed.
- Janeways, Immunobiology 8th ed.
Adaptive response graphic
What are Mature Naïve B Cells?
Mature Naive B Cells
BCR: IgM, IgD, Iga & Igβ
Co-BCR: CD19, CD81 & CR2 (CD21)
HLA-Class II
CD40
CD20
B-2 cells can be divided into two major subsets:
Follicular B cells are re-circulating B cells: Majority
Marginal B cells reside in the spleen: blood-borne polysaccharide Ags
B-1 cells: Mucosa – limited Ag specificity
And of course, HLA Class I
Describe the migration of mature naïve B Cells.
Naïve B cells enter the lymph nodes across the high endothelium venules (HEV) in the cortex
- Slow down : L-selectin, CXCR5 (Follicular chemokine receptor)
- Stable arrest : LFA-1
Once in lymphoid tissue they migrate to primary follicles to sample antigens and receive survival signals from Follicular Dendritic Cells (FDCs)
Competition for Survival Signals
Too many B cells, not enough FDCs to provide survival signals
•Naïve B cells die within weeks in absence of antigen
B cells that do not encounter specific Ag leave the node through the lymphatics and travel down the chain to the next lymph node
Particulate antigen filters through the lymph draining through the sinuses of lymphoid tissues.
HEV : high endothelial venule
What are follicular dendritic cells?
- Antigens are retained and concentrated in the follicles by follicular dendritic cells (FDC).
- FDCs are not hematopoietic and do not process antigen or express MHC/HLA Class II. NOT AN APC.
- Express receptors for C3b (CR1) and IgG (FcgR).
- Concentrate unprocessed opsonized antigen for:
- naïve B cells to “sample” for activation.
- activated B cells selecting of highest affinity antibodies.
- Secrete cytokines for B cell recruitment, survival and differentiation.
Describe B cell activation
Response initiated by recognition of antigen by BCR specific for that antigenic epitope
Complete activation requires 2 signals
Activation can occur in a T-dependent or T-independent manner.
Antigen dependent:
Th responses for second signal for protein antigens (T dependent)
Second signal from overwhelming BCR linkage mainly long repeating epitopes like lipids and/or saccharides (T independent)
What is the first signal of B cell activation?
First signal:
- Crosslinking of several BCRs with signaling through Iga and Igb ITAMs.
- Crosslinking of BCR with Co-BCR with signaling through Iga and Igb ITAMs and CR2 and CD19 signaling motifs.
- Crosslinking of BCR with TLRs with signaling through Iga and Igb ITAMs and TLR signaling motifs.
And of course, all three can be happening at the same time or two of the three, etc.
Describe what happens in B Cell Activation: First Signal
Crosslinking BCR signaling.
- Ag binding by mIgs.
- Must crosslink 2 or more BCR for signaling to occur.
- Signaling occurs through ITAMs on Iga & Igb cytoplasmic tails
- Syk – B cell phosphorylation
- The intracellular signaling steps in B- cell activation are identical to those of T cells. The only difference lies in the kinase involved in the initial intracellular signaling steps.
Describe what happens during B Cell Activation: First Signal
CO-BCR signaling.
CO-BCR: CR2, CD19, CD81
- Cross-linking of BCR by antigen generates a signal that is necessary but NOT sufficient to activate naïve B cells.
- Ag with bound C3d recognized by mIgs & CR2. (opsonized)
- CR2 provides cross-linkage for signaling.
- Signaling occurs through Iga & Igb, CR2 & CD19 cytoplasmic tails.
- If C3d is attached to protein Ag, Ag is ~1000 fold more immunogenic.
CR2 (CD21) : complement receptor 2
CD19 : pan B cell marker
CD81 : TAPA-1 -target of anti-proliferative antibody-1
Describe what happens in B Cell Activation: First Signal
Toll Like Receptor signaling.
Cross-linking of BCR by antigen generates a signal that is necessary but NOT sufficient to activate naïve B cells
TLR signaling:
Innate influence on adaptive response
Binding of antigen to both BCR and TLR.
Binding of PAMP leads to TLR signaling through cytoplasmic domains.
Amplifies the signaling response of Iga and Igb
Almost all TLRs are expressed on the surface of B cells and as well as cytosolic TLRs. Expression changes dependent on cell maturation and other factors.
You are not expected to know all the TLRs and when they ae differentially expressed for the purposes of this content.
What are the Outcomes of the First Signal of B Cell activation?
“Receptive “ B cell
- Prepares the cell for interaction with 2nd signal from Th cells
- Receptor mediated endocytosis of BCR and antigen
- Processing and presentation of Ag with MHC/HLA Class II
- Biochemical signaling
- Increased expression of cytokine receptors
- Secretion of low levels of IgM
What happens after activation of B cells?
Migration of Activated B Cells
After activation, B cells change their chemokine receptor expression and migrate to the edge of the follicular zone for interaction with activated Th cells for second signal. The newly activated T cells are also changing their chemokine receptors and moving towards the edge of the follicular zone.
B cells : downregulate CXCR5 and upregulate CCR7, migrate towards the paracortex, increase expression of HLA Class II and B7 (CD80)
Newly activated Th cells : Downregulate CCR7 and upregulate CXCR5, migrate towards follicles, increased expression of CD40L and cytokine secretion
What are the two pathways of the second signal of B cell activation?
Mitogen and Contact Dependent
Dependent on antigenic composition.
TD : T dependent. Proteins
TI : T independent - all the others. Mainly long repeating epitopes to be able to cross-linking several hundred BCRs to provide strong enough signal to by pass second signal from T cells
What is a T- dependent Immunological synapse?
All the same molecular players for B cell acting as an antigen presenting cell.
CTLA-4 : Cytotoxic T lymphocyte associated protein - 4
PD-1 : Programmed cell death protein 1
What happens during the B Cell Activation
T dependent Second signal?
2nd signal
- Activated CD4+Th cell recognizes antigen displayed by B cell within MHC/HLA Class II.
- B7 on B cell binds to CD28 on T cell
- CD40L on T cell binds to CD40 on B cell.
- Cytokines bind to cytokine receptors on B cell.
- Induced expression of activation-induced deaminase (AID) enzyme
- Proliferation and clonal expansion
Why does B cell Activation have a Two Pronged Approach?
CLONAL SELECTION
First signal : Recognition of antigenic epitope by BCR
Second signals: Maintains the specificity of the response to the specific epitope
This results in a large number of antigen-specific plasma cells and antibodies from rare antigen-specific naïve B cell.
What is induction of anergy?
B cells recognizing antigen without BCR cross-linking, binding of co-stimulatory ligands or cytokine support will not become activated.
These cells become unresponsive to additional stimulus
- anergic
- tolerant
Describe Migration of Activated B Cells- Additional diversity
After second signal from T cells, B cells change their chemokine receptor expression and migrate to the follicular area and establish germinal centers in the follicles. CCR7 (goes DOWN) CXCR5 (goes UP)
B cells begin cytokine modulated isotype (class) switching and affinity maturation (somatic hypermutation) of BCR genes.
Successful re-arrangements are selected/supported by TFH and follicular dendritic cells.
Tfh : T follicular helper cells , CD4+
What are T Follicular Helpers?
Tfh
- CD4+/low levels of CD25 expression
- Secrete IL-21, facilitates B cell survival, clonal expansion and differentiation to plasma cells
- Secrete Th cytokines to influence isotype switching
- Continued CD40L binding for B cell induction of AID
PD-1 : programmed cell death protein -1
CD95 : Fas
ICOS : inducible T cell co-stimulator
Describe Class Switching in the Germinal Center
Cytokines released by Th and Tfh cells promote two general functions.
The first is to induce heavy (H) chain class switching. They do this by opening switch regions in the heavy chain gene for somatic recombination.
The second function is to augment B cell differentiation and proliferation into plasma cells.
There is great redundancy in this system as many cytokines have overlapping functions.
Understand the type 1/type 2 cytokine influence on antibody class switching. You do not need to specifically memorize this table. You will need to know what the different Th cytokine profiles lead to as a predominate class switch.
Describe Heavy chain isotype switching
T-dependent Ags only : CD40/CD40L interaction is critical
CD40:CD40L ligation and cytokines trigger isotype switching and affinity maturation by:
- Opening of cytokine defined switch regions upstream of heavy chain constant regions
- Expression of activation-induced cytidine deaminase (AID) enzyme
VDJ gene segment recombines with a downstream C region gene and the intervening DNA is deleted
Normal DNA repair enzymes
This additional level of diversity only happens in B cells.
Describe Affinity Maturation : AID
T-dependent Ags only : CD40/CD40L interaction is critical
Introduction of point mutations in switch regions of variable areas of the heavy and light Ig genes resulting generation high-affinity antigen-specific antibodies.
Somatic Hypermutation: 103 to 106 times higher than normal spontaneous mutation rates
Key enzyme: AID, coverts Cs to Us
Mutations can be useful, sometimes not.
Isotype switching and affinity maturation often occur at the same time.
True or False: Isotype switching and affinity maturation often occur at the same time.
TRUE. Isotype switching and affinity maturation often occur at the same time.
Describe High affinity Selection Checkpoint by FDC
Selective survival of the B cells producing the highest affinity Abs occurs in the germinal center(s)
FDC and TFH are interactions with high affinity B Cells is necessary for survival
FDCs provide intact antigen to the new BCR receptor specificity to sample
If binds with a higher affinity, the B clone is selected for further differentiation to a plasma or memory cell
What are plasma cells?
Terminally differentiated effector B cell
- Short versus long-lived
- Cell markers:
- Decrease CD19 & 20, HLA Class II
- Increase CD27
- sIg class dependent – BCR is isotype switched to during process
- Secrete Abs at rates ranging from hundreds to thousands of antibodies per second per cell
- Long lived : bone marrow (IgG) and mucosa (IgA)
Abs: effector molecules of humoral immunity
What does the sIg is changed mean? It means the BCR is now a membrane bound IgG, IgA, or IgE isotype instead of IgM and IgD. The gene segments coding for M and D heavy chains have been cut out the gene and they cannot be expressed anymore. The new isotype will be expressed on the surface of the plasma cell or memory B cell.
What are memory b cells?
- Survive for long periods of time without additional Ag stimulation
- Express high levels of the anti-apoptotic protein Bcl-2, which contributes to their long life span
- Surface markers: CD 27, CD45R(O)
- sIg class dependent
- Need survival signals in the form of cytokine support
- Capable of mounting a rapid response to subsequent exposure to same Ag. (Secondary immune response)
- Easily differentiate into plasma blasts upon activation
What does the sIg is changed mean? It means the BCR is now a membrane bound IgG, IgA, or IgE isotype instead of IgM and IgD. The gene segments coding for M and D heavy chains have been cut out the gene and they cannot be expressed anymore. The new isotype will be expressed on the surface of the plasma cell or memory B cell.
Describe T-independent B cell activation
- Cross linking and clustering of several hundreds of BCRs
- Mainly seen with long repeating epitopes like polysaccharides
- Enough BCRs engaged to trigger enough Iga and Igb intracellular signaling to stimulate cell to secrete IgM.
- No class switching
- No affinity maturation
- Little to no memory
- Can provide IgM antibody protection in a short amount of time
Describe antibody feedback
•Control mechanism triggered by excess secreted Ab bound to a pathogen that blocks further Ab production.
- Immune complexes majority – unbound pathogen minority
- Same pathogen that activated the cell
- Immune complex binds to Fc inhibitory receptor on B cells: FcgRIIB
- FcgRIIB contains ITIM to block further signaling and activation of the B cell.
- IgG only
-Unbound IgG cannot bind to the receptor
If Ab is in excess, it’s means it’s not needed to clear a pathogen. The threat has been handled and now contraction of the immune response can occur.
What happens to the long-lived plasma and memory cells?
•Long-lived plasma and memory cells migrate to survival niches.
- Bone marrow
- Mucosal lymphoid tissues
- Response to next challenge is secondary response. Third is tertiary response. And on.
- Each activation leads to higher affinity antibodies and larger population of memory and long-lived plasma cells being generated.
What are Antibody Effector Functions?
Isotype switching diversifies functional properties
Fc region serves two distinct functions
◦Deliver antibody to inaccessible anatomical sites
◦ Link bound antigen to molecules/cells that effect destruction – OPSONIN
Fc region contains binding sites Fc receptors (FcR) : IgG and IgE
Binding sites for complement : IgG and IgM
Describe Fc RECEPTORS ON IMMUNE CELLS.
Immune complex (IC) binding via Fc receptor on phagocytes leads to receptor endocytosis and antigen processing
•Binding of IC to FcR acts through signal transduction complexes to alter gene expression in cells (increases activation)
Immune complex : antigen bound to antibody. Can be any antibody isotype.
Describe the FcRn RECEPTOR FOR IgG.
- FcRn – n is for neonatal
- IgG specific
- Functions in recycling and transcytosis of IgG in a pH dependent manner
- Important in maintaining IgG levels in the serum.
- Increases the half life of IgG by protection from proteolysis
- Expressed on :
- APCs
- Neutrophils
- Vascular endothelium
- Mucosal epithelial cells
- Podocytes
What are the Effector Functions?
Describe Neutralization of microbes and toxins
Antibodies neutralize the infectivity & potential effects of infection. Many microbes and toxins bind to the surfaces of cells to gain entry.
IC sterically hinder binding of microbes and toxins.
Only requires binding to Ag.
Any class of Ab works.
The higher the affinity, the better.
Known as “neutralizing Abs” or “blocking Abs”.
Important in viral infections that spread via extracellular mechanisms
Describe Opsonization & Phagocytosis of microbes
Antibody mediated opsonization
- Binding of ICs to Fc receptors on immune cell populations promotes phagocytosis and increases activation of the phagocyte.
- Increased killing enzyme production and ROS bursts to degrade pathogen.
- Increased expression of co-stimulatory ligands and cytokines for Th cell interactions
Describe Complement activation
Classical Pathway
Antibody-directed mechanism of activation
- Most rapid and efficient pathway
- Initiation of pathway: IgG (2) or IgM
- Fc conformational changes exposes C1q complement receptor (CR)
- IgM staple configuration exposes all five C1Rs (efficient complement “fixer”)
- At least two molecules of IgG must be bound in close enough proximity for C1 to bind multiple globular domains of C1q between to activate enzymes
C1 component
-one C1q subunit (hexamer) associated to two C1r and two C1s molecules, large complex
Describe ‘Three pathways one end point’
- All three C’ pathways end with the polymerization of many C9s for formation of the membrane attack complex (MAC).
- All three pathways can be occurring at the same time (antigen dependent).
What are Cooperative interactions?
- IgG and C3b opsonin mediated phagocytosis.
- Pathogen bound by antibody and C’ components are much more readily phagocytosed and stimulate phagocytic killing mechanisms
- Stronger phagocytes responses than just C3b or IgG alone
Describe Waste Management re: immunoglobulins
- Opsonization with IgG and C3b allows for clearance of immune complexes (IC) from circulation in a non-inflammatory manner.
- CR1 on erythrocytes binds circulating immune complexes with attached C3b and C4b and transports the complexes to the liver and spleen.
- Organ resident phagocytes remove the immune complexes from the erythrocyte surface and the erythrocytes continue to circulate.
- Phagocytes degrade the ICs
ADCC: Anti-viral state
- IgG binds to surface bound antigens
- NK cells bind to antibody-coated cells by Fc receptors
- Degranulation of NK cell Perforin and granzyme granules
- Induced apoptosis of target cell
Describe Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC).
- NK cells
- Macrophages
- Neutrophils
- Eosinophils
- Targeted recognition through IgG or IgE binding to surface antigens.
- FcR on immune cells bind to antibody activating cell killing mechanism(s).
- Killing by lytic enzymes, TNF and Perforin/ granzyme
Describe IgE Mediated Reactions:
Coming Attraction
- Th2 cytokines influence class switching to IgE.
- IgE binds to FcεRs on present on mast cells and eosinophils.
- Ag binding causes degranulation of cell.
- Granular content is specific for defense against helminths
- Type I hypersensitivity
Describe Mucosal Immunity: Coming Attraction
- MALT / GALT
- Protection at the most common portals of entrance.
- Non-inflammatory
- IgA: Secretory antibody
- J chain and secretory peptide
What are natural antibodies?
We make “naturally” occurring antibodies to blood group antigens we don’t have on our RBCs
Mainly IgM and IgG, ~ some IgA
Produced by B-1 and marginal zone B cells in the MALT and Spleen
•Exposure to intestinal bacterial, viral and/or food antigens.
May play role in:
- Autoimmune diseases
- Inflammatory diseases
- Immune homeostasis
Describe Maternal-Fetal-neonate Immunological Protection
During pregnancy, the fetus receives IgG from the maternal circulation
Additional protection provided by maternal IgA secreted in breast milk after birth
IgG only class of Ab to cross placenta. IgA major class of antibody in secretions.
Describe FcRn: IgG Transport Receptor
FcRn: transports maternal IgG from across the placental barrier into fetal circulation.
IgG can leave circulation and enter extracellular spaces within tissues via transcytosis mediated by FcRn (FcRb).
Maternal IgG is pinocytosed and bound to FcRn in the endosomes.
FcRn targets bound IgG for endosome tranlocation to the fetal circulation where it is released at neutral pH.
IgG only
-Provides broad protection
FcRn – Fc receptor natal IgG only class of Ab with this type of receptor
The First Year is the Hardest-Immunologically! Why tho
After birth, the remaining maternally derived serum IgG degrades over a few months
IgM and IgA are produced a short time prior to birth but not in high quantities
IgG is produced after birth
All have a lag time for developing protective quantities in the serum leaving a vulnerable period from around 6-12 months of age when a baby is not completely immunocompetent
Describe Monoclonal antibody therapeutics
- Highly specific recognition of epitope
- Very high affinity
- Stimulate a variety of immune responses
- Side effects
- Can produce large quantities quickly
- Expensive
- Cancers
- Autoimmune diseases
Describe Evasion of Humoral Immunity
- Pathogens have evolved many mechanisms for evasion of humoral responses.
- Change of surface Ags.
- Outer capsule or coat prevents complement activation.
- Capsule prevents binding of Abs and phagocytosis
