LECT 4.

1 NOTES: B CELL EFFECTOR FUNCTION

Key QN: How do B cells execute their effector functions?
1] Memory B Cell VS Plasma B Cell
RECALL: Germinal Center Formation and Function:
Germinal center contains dark zone and light zone
⇒ B cell activated, undergo proliferation and acquire mutations
⇒ Introduce mutations into genes coding for BCR (Somatic hypermutation, AID to introduce mutations)
⇒ Change binding affinity to antigen
● If they lose affinity ⇒ Undergo apoptosis (death)
● If they gain affinity ⇒ Positively selected, undergo affinity maturation

What happens if the affinity of BCR towards antigen in the initial activation is not high enough?
Ans: Not all activated B cells undergo germinal center reaction (for EG: Follicular B-2 cells)

T cell-dependent memory B cell generation (GC-dependent and independent)

B cells which do not have high affinity of BCR are activated but insufficient to go into germinal center reaction
⇒ Exit follicles, become short-lived plasma cells (b)

B cell does not undergo somatic hypermutation + class-switching

● Usually stay as IgM isotope
● Not as high affinity to antigen but can produce antibodies quickly
⇒ Forms memory B cells (c)

B cell has higher affinity

● Undergo germinal center reaction (d)
● Undergo clonal expansion, acquire mutations
● Enter light zone, selected against antigens
⇒ High affinity B cells able to engulf more antigen, produce more peptides, present more to T cells which produce
cytokines to help B cells survive better (advantage)
How B cells decide to become Memory/ Plasma B cells

Very high affinity B cells at stage (d):

● Receive a lot of T cell help, tend to become long-lived plasma cells (f)
● Its antibody produced can bind to antigen with high affinity
● Reside in Bone Marrow (BM)

High affinity BCR (but not as high as previous one)

● Receive less T cell help, tend to become memory B cells (e)
● Repeat encounter with pathogen, pathogen might be slightly different from previous one
● If its antibody is not good for the new pathogen, Memory B cells with different affinity activated, undergo further
mutation to become high affinity B cells ⇒ Become plasma cells, produce perfect antibody

KEY CONCEPT: Once B cells become plasma cells, they ONLY produce 1 specific type of antibody (No longer has the
cell surface receptor) VS Memory B cells which has BCR, can still respond to antigen binding and change their affinity

During GC reaction, how do GC B cells decide to go back to dark zone? (No need memorise this part)
Ans: Determined by chemokines (Different in light and dark zones)

Dark zone:
CXCL12 production, corresponding CXCR4 receptor

Light zone:
CXCL13 production, corresponding CXCR5

When B cell receptor have high affinity to antigen BUT not

high enough to become memory B cells / plasma cells
⇒ Signal not strong enough, downregulate CXCR5 and
upregulate CXCR4 to be recruited to dark zone again
⇒ Engage extension process, acquire more somatic
hypermutation, selected again in light zone
T-independent B cell activation:
● Simple, repetitive epitopes (often carbohydrates)
● Mostly IgM (produced short lived plasma cells)
● Modest affinity (No SHM)
● If IFNγ is induced IgG3, IgG2a can be produced as well
● No memory
● B cells activated by direct BCR crosslinking
● B cells can also be activated via Toll-like receptors (TLRs) ⇒ Pathogen recognition receptors

Most B1 cells and marginal zone B cells activated through this pathway to become plasma cells
⇒ Mostly produce IgM antibodies

2] Antibody secretion

Both B cell receptor (BCR) and antibodies are encoded by the same locus, why one is membrane bound and the
other is secreted?

How to produce secreted Ig instead of membrane bound Ig?

Constant region (Eg. Of IgM) ⇒ Encoded by 6 exons

● Last 2 exons encoding for transmembrane domain; Secretion domain behind 4th exon; PolyA site behind 4th
exon and transmembrane domain exon
● Membrane-bound IgM uses the polyA site behind the transmembrane domain exons
⇒ Produces mRNA which contains sequence coding for transmembrane domain
● Secreted IgM uses the polyA site behind the secretion domain
⇒ mRNA contains sequence for secretion domain ⇒ Protein produced secreted outside cell
3] Antibody effector functions
Why do we need to produce antibodies of different isotypes?

● Transport across epithelium → Major class IgA

● Transport across placenta → Major class IgG1
● Diffusion into extravascular sites → All IgG subclasses (Small enough to diffuse)
● Present in different concentration → In serum, most abundant is IgG1, least is IgE
● IgA is most abundant in the body → Found in all mucocyte area (Large intestinal tract + Respiratory tract)

Antibody functions

Neutralisation

Block interactions b/w

bacterium/ virus and a
cell surface receptor

Pathogens cannot just enter your cell ⇒ Depends on binding to surface receptor
● Antibodies which can bind to surface of receptor / pathogen prevents binding
● Mainly IgG subclasses and IgA present ⇒ First line of defence mucosa
● Complement response mainly involves IgM and IgG3
Opsonisation Opsonization involves the binding of antibody to the pathogen
● Antibody forms a coating surrounding the pathogen due to recognition
Process by which a
pathogen is marked for ● Fc portion (constant region of antibody) binds to the Fc receptor on the
ingestion and destruction phagocyte cell surface membrane → sends signal into phagocyte.
by a phagocyte
● Phagocyte engulfs antibody together with pathogen
⇒ Speeds up the process

Sensitisation Mediated by IgG1 and IgG3

for NK cell killing

Antibody-Dependent
Cell-Mediated
Cytotoxicity (ADCC):

Large parasite, too big for any immune cell to engulf

→ Only way to eliminate pathogen is to destruct them
→ Antibody recognises antigen, antibodies bind to Fc receptors on surface of NK cell
→ Fc receptors cross-linked and activated
→ NK cells produce perforin, granzyme and other inflammatory agents to destroy
pathogen
→ Pathogen lysed

Sensitisation Mediated mainly by IgE

of mast cells

NOTE:
Mast cells, eosinophils and
basophils are all involved in
defence against parasites

● Eosinophil and Basophils also express FcεRI, but located at different places +
have slightly different functional profiles
● Basophils (1%) and eosinophils (2.3%) circulate as mature cell types with short
half-lives
● Mast cells exist only in tissues
● Eosinophils express a different rang of granule contents to mast cells and
basophils
Locations of antibody isotypes

● IgG and IgM mainly found in blood circulation

● IgG and monomeric IgA relatively small, able to diffuse

into extracellular body

● IgG1 found in foetus

● IgE reside in tissues underneath epithelial surface

● IgG and dimeric IgA found in breast milk, respiratory and

intestinal tracts

Why are IgM and IgD expressed on the same cell at the same time, but not other isotypes?

Staining of splenocytes with IgM and IgD:

[1] Premature cells first arrive in the spleen, expressing only IgM
[2] Greatly upregulate IgD expression. Maturation will downregulate IgM expression.
[3] Mature cells mostly express IgD

Reason why splenocytes can express BOTH isotypes:

In the constant region, ONLY μ and δ controlled by 1 switch region, the rest have their
own switch regions. After transcription, alternative splicing creates either IgM or IgD

If switched to another isotype (Eg. IgG1), no longer have other isotype ⇒ μ, δ and γ3 portions already spliced out
⇒ Only can express IgG1 (First isotype)
● Cannot express isotypes before this site ⇒ Already spliced out
● Cannot express isotypes after due to polyA site ⇒ Transcription terminated
4] Fc receptors

Fc receptor helps to transport IgA antibody across mucosal epithelial cells

[1] IgA forms dimeric structure

[2] Held together by J-chain

(important for stabilisation of antibodies since
monomers are less stable than dimers +
[3] recognised by polymeric Ig receptor for
[4] binding and internalisation into mucosal
epithelial cells)

⇒ [5] IgA transported across epithelial cell into

lumen, IgA released when polymeric Ig receptor is
cleaved
⇒ Portion of polymeric Ig receptor remains bound
to IgA

FcRn mediates the transfer of IgG during pregnancy and in newborn baby:

When newborn baby ingests milk, it IgG present in maternal blood

contains IgG antibodies produced by
mother → Absorbed into syncytiotrophoblast
by endocytosis
→ IgG binds neonatal receptor in
intestinal tract (Lower pH value) → Early endosome has acidic
condition, binds to neonatal receptors
→ Engulfed into epithelial cells, and transported to other side
transported across the cell
→ pH reaches neutral, antibody
→ Reach neutral pH condition, IgG released into foetus
released
 LECT 4.2 NOTES: T CELL DEVELOPMENT
Key QN: How are T cells generated and selected?
● T cells are developed and matured in the thymus (unlike B cells which are still immature upon leaving the BM)
● Age-related thymic involution (thymus is the only organ that shrinks as we grow older)

Old age: thymus is not detectable, filled with fat only ⇒ Do not produce new T cells with unique T cell receptors

2 distinct lineages of immature T cells:

● αβ (major population) → develops into CD4 and CD8 T cells
● γδ (minor population)

1] T cell development

Developing αβ T cells:
Most important markers of αβ T cells: CD4 and CD8

Double Negative Cells (CD4-, CD8-)

DN1 stage Earliest T cell precursors in thymus express CD44 ONLY

DN2 stage Upregulate CD25

Transition from DN2 to DN3 stage:
● Start to prepare their TCRβ locus (Similar to antibody heavy chain)
● Undergo VDJ recombination to generate TCR

DN3 stage Downregulate CD44, become CD25 single positive only

DN4 stage Start to upregulate CD4 and CD8 expression

Cells undergo TCRα chain recombination (Similar to light chain)

Double Positive Cells (CD4+, CD8+)

● With maturation, becomes single positive ⇒ Either CD4 or CD8 cells
● Undergo +ve / -ve selection ⇒ Becomes single positive ⇒ Single positive cell undergoes -ve selection to
ensure all cells passing through this stage are not autoreactive (Process to eliminate autoreactive T cells)
2] TCR V(D)J recombination
TCR loci for α, β, γ, δ

TCR β chain locus

● More similar to heavy chain. Contain V, D and J segments but with slightly different arrangement
● B cells contain a cluster of V segment, cluster of D segments and cluster of J segments;
T cells contain J segments which has their own D segment
● GENE ARRANGEMENT:

TCR α chain locus

● More similar to light chain. Contain V and J segments ⇒ Recombination occurs in just 1 step
● GENE ARRANGEMENT:

● TCR δ chain locus is embedded within the α locus

○ Contains V, D and J segments
○ Even with recombination of δ locus, if α locus recombination occurs, entire TCR δ locus is lost
TCR Somatic Recombination (how this all makes the TCR)

Recombined regions (VDJ from β, VJ from α) form

the antigen-binding domain
⇒ creates diversity in TCRs

Processes in B cells also exist in T cells:

● Imprecise V(D)J end joining (Not precise)
⇒ Deletion, P- and N-nucleotide additions
(all joining processes)
● TdT (Enzyme generating N-nucleotide)
is expressed in all T cell precursors
● Allelic exclusion ⇒ Less tight, similar to light
chain ~1-10% inclusion
● No somatic hypermutation

Flowcytometry analysis of developing T cell in the thymus

3] TCR vs BCR

Pre-TCR VS TCR Pre-BCR VS BCR

● Pre-TCR and TCR associated with CD3 receptors (5 Pre-BCR and BCR both associated with Igα/β
different chains) ⇒ Co-stimulatory co-receptors which aid in receptor
● Pre-TCR stage, β chain already recombined but α signalling
chain not ready
⇒ Additional chain pTα which encode protein that
associated with β chain without need for
recombination (Similar to VpreB and lambda 5 for
pre-B cell receptors)

Antigen binding differences

Antigen bind to TCR Antigen bind to TCR

TCR can only recognise antigen in peptide form BCR can bind directly to antigen
(presented by MHC molecule) due to small binding region
⇒ TCR binds unstably to MHC
⇒ require coreceptor help (CD4 for MHC Class II, CD8 for
MHC Class I)

Similar downstream signal for BCR and TCR

● Firstly activates Src family kinases ( [1] TCR activates Lck/Fyn; [1] BCR activates Lyn/Blk)
● Activate secondary kinases ( [2] ZAP 70 in T cells, [2] SYK in B cells)

T cells need to be exposed to antigens in different stages of its maturation.

● During early stage selection in thymus: MHC peptide presented on thymic epithelial cells (TECs) and DCs
● DUring functional matured state: MHC peptide presented on APCs
4] Thymocyte selection (way more complicated than B cell)

[1] T cell precursors generated in bone marrow, enter thymus at border between 2 domains

[2] Become DN1, DN2 and DN3 cells

[3] DN3 cells complete β recombination, undergo β selection

[4] Between DN2 and DN3, γδ T cells generated

[5] DN4 stage. Upregulate CD4 and CD8 to give DP cells.

[6] DP cells undergo positive selection against peptide presented by cortical thymic epithelial cells (cTEC)
Purpose: to select T cells which can recognise your own (self) MHC molecules, otherwise eliminated by apoptosis

[7] Pass selection process, become SP cells. Moves to medulla.

[8] SP cells undergo negative selection by peptide presented by medullary thymic epithelial cells (mTEC) + DCs
Purpose: to select T cells which do not recognise self peptide / MHC, otherwise eliminated by apoptosis
(However, for cells that still recognise MHC but do not bind strongly ⇒ pass negative selection and become mature T cells)

5] Details on step 7 & 8

Step [7]: CD4/ CD8 lineage decision (what happens during DP to SP transition)

DP cells express BOTH CD4 and CD8 coreceptors

⇒ can engage both Class I and II MHC
⇒ if the cell is better matching to MHC II (for eg.) means there are difficulties binding to MHC I molecules due to
structural differences
⇒ Class II binding more useful, CD8 downregulated
⇒ Selected into CD4 or CD8 cell based on binding affinity to class II peptide
⇒ Become CD4 single positive cell, undergo negative selection in medulla
Step [8]: SP cells undergo negative selection in medulla by mTECs and DCs

Central tolerance: Delete T cells with TCR recognizing tissue antigen derived peptide presented on MHC
⇒ Negative selection process

mTEC
● Expresses specific TFs (Aire and Fezf2)
● Promote expression of tissue-restricted antigens (TRAs)
● Only a few cell types like mTEC are present in thymus. Other tissues (eg. skin, liver) don’t express self-antigens
● Antigens processed and presented on MHC molecules for T cell selection

Dendritic cells
● Able to uptake tissue-specific antigen produced by mTEC and present it on MHC Class II molecules
⇒ Contribute to Class II selection through presentation
● A lot of CD4 Treg cells created like that via the MHC II pathway
→ this DC mechanism plays a major role in T cell regulation

Recall this concept of B cells being in different stages:

Immature stage ⇒ B cells which have strong binding to self antigen undergo apoptosis;
But once mature, B cells have different internal composition and become activated upon binding to antigen rather than die

Similar concept with the T cells:

● Strong binding ⇒ apoptosis
● Binding with intermediate affinity ⇒ Regulatory T cell, (if Upregulate Foxp3 ⇒ inhibitory T cells)
● Weak binding to antigen ⇒ conventional T cells
● Dendritic cells able to uptake tissue-specific antigen produced by mTEC and present it on MHC Class II
molecules ⇒ Contribute to Class II selection through presentation
6] MHC antigen presentation is also required for the positive and negative selection in the thymus

MHC antigen presentation pathways

Class I presents endogenous antigen

⇒ Species infected by pathogens / produced
endogenously
⇒ Processed by proteasome into peptide,
loaded onto Class I molecule with help from
association molecules
⇒ Expressed on cell surface, can engage
CD8+ cells

Class II presents exogenous antigen

⇒ Engulfed into endosome with many class II
molecules, loaded and presented on cell
surface to CD4+ T cells

MHC antigen presentation for positive and negative selection in the thymus
Positive selection ⇒ ONLY have self antigens, NO foreign antigens

Scenario 1: DP ⇒ CD8SP
cTEC: Endogenous peptides processed by thymoproteasomes (ONLY expressed in cTECs)

Scenario 2: DP ⇒ CD4SP
● Problematic, do not have anything which can be engulfed through endocytosis
⇒ Everything presented cTEC is produced exogenously
● In order to present exogenous peptides only Class II
⇒ Autophagy-dependent loading of endogenous peptides for Class II presentation in cTECs

Standard-, immuno- and thymo-proteasomes:

Most cells express standard proteasome

Most immune cells express immunoproteasome,
thymoproteosome expressed in cTECs

Immunoproteosome, subunits become 1i, 2i, 5i compared to regular proteasome

Thymoproteosome only replaces last subunit 5i with 5t (Unique subunit)
→ if 5t subunit not there, this will result in defective CD8+ SP cell differentiation (+ve and -ve selection at the same time)

Why we need thymoproteasome?

Different peptides for +ve and -ve selection, even if they are derived from the same antigen
Summary of MHC antigen presentation for positive and negative selection in the thymus

7] T Cell differentiation?

How do mature T cells leave the thymus

50% of T cells leave via the blood, 50% of T cells leave via the lymphatic vessels (Most B cells go through the blood)

Mature naïve T cells go to lymph nodes or spleen ⇒ Activated by dendritic cells (or B cells)

B and T cells enter lymph nodes through artery; T cell can also enter via lymphatic vessels
⇒ T cells go to T cell zones, B cell go to B cell zone forming germinal centers

T cell priming by dendritic cells (2 steps)

Step 1: Engagement of antigen presented by migrated dendritic cells (From skin / other periphery tissues) which enter
the lymph node ⇒ Activate either CD4 and CD8 T cells
Step 2: Another tissue specific LN-resident dendritic cell able to activate BOTH CD4 and CD8 T cells at the same time
⇒ CD4 can help fully activate CD8 T cells ⇒ Increase cell number, cytotoxicity and migrate to infection site
 8] CD4 T cell subsets → Effector cell OR Regulatory cell

Upon activation ⇒ Polarisation of CD4 T cells in the periphery:

Clonal expansion (T cells) Effector function:

T cells encounter Microenvironment produces cytokines (By NK cells, macrophages or dendritic cells)
intracellular pathogens ⇒ Cytokines promote Th1 differentiation
(bacteria, virus) ⇒ Produce effector cytokines which affect macrophages involved in activation CD8 and NK cells
(Th1 type response)

T cells encounter IL-4 mostly produced by NK cells

Extracellular pathogens ⇒ Th2 differentiation
(bacteria, parasites) ⇒ Cytokines produced by Th2 cells involved in activation of B cells and activation of macrophage
(Th2 type response) in alternative way (Involved in tissue regeneration rather than inflammatory response)

T cells encounter ⇒ TGFβ and IL-6 produced

Extracellular pathogens ⇒ Th17 differentiation
(bacteria, fungi) ⇒ IL-17 involved in activation + recruitment of neutrophils and monocytes
(Th17 type response)
Th1 type immune response (against intracellular pathogens)

Th2 type immune response (against extracellular bacteria and parasites)

B cell perspective
Th17 type immune response (against extracellular bacteria & fungus)

Treg cells

Once most of infection cleared, no more IL-6 produced, only TGFβ remaining
⇒ Promote generation of Treg cells ⇒ Produce inhibitory cytokines (IL-10)

Natural Treg (nTreg) and induced Treg (iTreg)

Natural Treg cells produced via negative selection in the thymus

Induced Treg cells generated upon antigen stimulation
⇒ Induced after infection has been under certain control, want to reduce immune response
⇒ Additional Treg cells that don’t express Foxp3, just produce IL-10 ⇒ Tr1 and Th3 (Represent MOST of the iTreg cells)
To verify that Tregs have suppressive function (do in vitro suppression assay)
Basically incubate purified T cells labelled with dye staining plasma membrane.
Plate 1: T cells only // Plate 2: T cells with Treg cells.

Stimulate T cells in both plates with TCR, Anti-CD3/CD28 stimulation

● Plate 1: T cells continue dividing, dilution of membrane dye ⇒ Fluorescence level drops
● Plate 2: T cells don't divide due to Treg suppressive effect ⇒ Fluorescence level remains high

9] CD8 T cell subsets →Tc1, Tc2

CD8 T cells also differentiate into different subsets upon activation (depending on cytokines produced)
● Tc1 and Tc2 are major subsets, have higher cytotoxic activity (most potent killer) which apart from killing
infected cells directly, can produce cytokines which further modulate host immune response
● Tc2 cells have slightly weaker cytotoxic activity than Tc1 ⇒ Cytokines produced help Helper T cells