Lecture 5: T-Cell and B-Cell Activation, Differentiation and Memory

Question 1

Briefly describe the steps involved in the activation of the adaptive immune response

Answer 1

• APCs activated by their PRRs binding to PAMPs at the site of infection
• processed antigens displayed as peptides on surface of APC in groove of MHC class I and II molecules
• at the same time, these APCs have migrated to secondary lymphoid organs (lymph nodes/spleen)
• APC interacts with naive T-cell to activate the adaptive immune response

Question 2

What is T-cell recirculation?

Answer 2

Naive T-cells continually re-circulate between the blood, lymphatics and secondary lymphoid organs
- when enters a secondary lymphoid organ, naive T-cells browse cells for APCs expressing a MHC/peptide complex to which to can recognise and bind to in order to initiate T-cell activation

Question 3

What is the purpose of T-cell re-circulation?

Answer 3

increases the probability of the T-cell finding its specific antigen

Question 4

Explain the two-signal hypothesis of T-cell activation

Answer 4

Costimulatory signals are required for optimal T-cell activation and proliferation

Signal 1: interaction of the TCR/CD3 complex with MHC/peptide complex

Signal 2: co-stimulatory signal via CD28 which recognises CD80/86 on APC

(also third signal being soluble cytokines to determine the type of T-cell response)

Question 5

True or False: CD80/CD86 is downregulated upon APC activation?

Answer 5

False: CD80/CD86 is upregulated upon APC activation to allow costimulatory activation of Native T-cell

Question 6

What is the name of the organisation of signalling molecules during successful T-cell-APC interaction?

Answer 6

immunological synapse

Question 7

Describe the organisation of the immunological synapse formed for successful T-cell - APC interaction

Answer 7

Forms a region known as a supramolecular activation complex (SMAC) with central (cSMAC) and peripheral (pSMAC) regions
cSMAC = TCR/CD3 complex - MHC interaction (with CD4/CD8)
within cSMAC but towards the periphery, there is the co-stimulatory CD28 of T-cell and CD80/86 of APC interacting
pSMAC = integrins on T-cell interact with receptors on APC to increase avidity of interaction (maintains signal into T-cell by physically holding the cells closer together)

Question 8

What is the role of the immunological synapse in T-cell-APC interaction?

Answer 8

to bring signalling molecules into close proximity and increase the chance of phosphorylation of signalling molecules and thus successful T-cell activation

Question 9

What are the positive co-stimulatory receptors/ligands?

Answer 9

Receptor CD28, which interacts with CD80/86 on APCS for initial T-cell activation

Receptor ICOS, which interacts with ICOS-L on APC for maintaining activation of differentiated T-cells and has roles in effector and memory T-cells

Question 10

What are the negative co-stimulatory receptors and what are their purpose?

Answer 10

Negative receptors are important for switching off T-cell responses in order to resume homeostasis
Receptors include: CTLA-4, PD1 and BTLA

Question 11

How does CTLA-4 T-cell receptor work to downregulate T-cell responses?

Answer 11

Negative co-stimulatory response:
CTLA-4 has stronger binding affinity for CD80/CD86 on APC so outcompetes CD28 to turn off stimulatory signal for T-cell activation.

Question 12

What happens when the co-stimulatory signals are not present for T-cell activation or when negative co-stimulatory response occurs?

Answer 12

MHC recognition in absence of co-stimulatory response
and
MHC recognition with negative co-stimulation
= both scenarios lead to anergy (T-cell turned off because one signal not enough to activate T-cell - Two-signal hypothesis)

Question 13

Which APCs are the best at activating Naive T-cells?

Answer 13

Dendritic cells (known as professional APCs)

Question 14

What drives the activation of macrophages for antigen presentation?

Answer 14

PAMPs (promote upregulation of CD80/86)
INF-gamma from T-cells (promote upregulation of MHCII)

Question 15

How are helper T-cells involved in B-cell activation

Answer 15

Resting B lymphocyte recognise antigen via BCR, causing internalisation, degradation and peptides displayed on MHCII
- B cells shows peptide to helper T-cell that has already been activated in response to same pathogen
- Helper T-cells release cytokines that signal to the B-cell to determine the type of antibodies made

Question 16

What are superantigens?

Answer 16

bypass the normal rules for TCR antigen specificity
- can simultaneously bind to the Vbeta regions of TCRs and the alpha chains of MHC class II non-specifically
- not antigen specific so can result in activation of too many T-cells that would not have been activated ordinarily

Question 17

Give 2 diseases related to exogenous superantigens

Answer 17

Toxic shock syndrome
Food poisoning

Question 18

what can result from T-cell activation by a superantigen?

Answer 18

polyclonal T-cell activation and dramatic cytokine release

Question 19

Describe how Native T-cell activation leads to T-cell differentiation

Answer 19

activation of naive T-cell increases secretion of cytokine IL-2 and upregulation of its receptor IL-2R/CD25 for autocrine stimulation that results in proliferation, production of memory T cells and clonal effector T-cells

Question 20

What is the role of polarizing cytokines (third signal) during T-cell activation?

Answer 20

determines the subset of T-helper cell differentiated
- depending on the PAMP and PRR signalling, APC will secrete different cytokines
- these cytokines signal to CD4+ T-cells via STAT signalling pathways and influence T-helper cell differentiation by activating transcription master regulators that turn on defined sets of effector cytokines

Question 21

Describe the activation of Type 1 helper T-cells and their effector function

Answer 21

In response to intracellular pathogens:
signal 1: MHC class II
signal 2: CD28-CD80/86
signal 3: PRR signalling in APC results in secretion of IL-12

IL-12 activates the STAT4 pathway in the naive T-helper cell, which activates master regulator T-bet that upregulates interferon gamma to activate macrophages to clear the intracellular pathogen

Question 22

Type 1 T-helper cells are important in which type of immunity?

Answer 22

cell-mediated (intracellular pathogens, cancer)

Question 23

Type 2 T-helper cells are important in which type of immunity?

Answer 23

humoral (multicellular pathogens, extracellular pathogens)

Question 24

Describe the activation of Type 2 helper T-cells and their effector function

Answer 24

In response to multicellular pathogens (E.g. worms):
signal 1: MHC class II
signal 2: CD28-CD80/86
signal 3: PRR signalling in APC results in secretion of IL-4

IL-4 activates the STAT6 pathway in the naive T-helper cell, which activates master regulator GATA-3 that upregulates IL-4, IL-5 and IL-13, which promote eosinophil activation in an allergy response and direct class-switching of B-cells to produce IGE antibodies

Question 25

What is cross-regulation in T-helper subset activation?

Answer 25

Depending on the polarizing signals received from the activated APC, either a type 1 immune response (with activation of master regulator T-bet) or a type 2 immune response (with activation of master regulator GATA-3) will be activated.
T-bet and GATA-3 inhibit each other ensure only one type of T helper cell is stimulated.

Question 26

Other than Type 1 and Type 2 T-helper cells, what other T-cell subsets can be produced by different polarizing cytokine signals?

Give 2 examples (but there are many others and depends on the pathogen encountered)

Answer 26

T-regulatory cells (important in turning off the T-cell response) = can be induced by TGF-beta and IL-2 to turn of master regulator FoxP3

T-follicular helper cell (important in directing germinal centre responses) = can be induced by IL-21 to turn of master regulator Bcl6

Question 27

What are the two main subsets of memory T-cells, where are they found and what is their role?

Answer 27

1. Central memory T-cells (Tcm) - found in secondary lymphoid organs, role in responding to reinfection by differentiating into T-helper cell subsets under cytokine influence
2. Effector memory T-cells (Tem) - found in periphery, maintained within the tissues and recalled faster upon reinfection

Question 28

where are B-cells activated?

Answer 28

follicles of secondary lymphoid organs

Question 29

What is the clonal selection hypothesis of B-cell activation and differentiation?

Answer 29

each B-cell has a single type of Ig receptor and upon stimulation, each cell will create a clone of cells bear the same Ig receptor as the original - the clones will have have the same specificity for the antigen as the original
- the clines become plasma cells that secrete antibodies or become memory cells

Question 30

What are the three modes of B-cell activation by B-cell antigens?

Answer 30

1. most B-cell antigens are T-cell dependent and need to be presented to T-helper cells so that the appropriate help can be delivered via cytokine signalling
   –> primarily B2 B-cells
2. some antigens are T-cell independent and do not require T-cell help is they can stimulate PRRs on B-cells
   –> these antigens are normally multivalent (pathogen has multiple copies of same antigen) binds to PRRs and multiple BCRs causes receptor clustering on B cell surface - strong signal
   –> more likely to activate B1 B-cell and MZ B-cells
3. Other antigens are T-cell independent and may fix complement fragment C3d and bind to the complement receptor CD21 on B-cells and BCR to increase the signal into the cell

Question 31

What are the T-dependent B-cell responses?

Answer 31

upon binding antigen via BCR, some antigen degraded and displayed on MHC class II, there is upregulation of CD40 co-stimulatory receptor and cytokine receptors.
Interaction with T-helper cells (antigen bound by TCR, CD4 recognises MHC class II and CD40L binds CD40 of B-cell)
–> This allow full activation of the B-cell, which undergoes clonal selection and expansion to:
- enter germinal centre reaction (leads to formation of memory cells, primary focus of high affinity long lived plasma cells)
- form a primary focus (low affinity short lived plasma cells)
- form memory cells.

Question 32

Are memory cells produced by T-independent B-cell activation? why/why not?

Answer 32

No because the formation of memory cells requires T-cell help.

Question 33

How do B-cells encounter antigens in the lymph nodes and spleen?

Answer 33

Small soluble antigens can enter the lymphatic system move into the lymph nodes via the afferent lymph - they are small enough to fit through the gaps between the epithelial cells within the subcapsular sinus into the follicles where they are captured by follicular B-cells

Larger antigens care captured by the subcapsular sinus macrophages (SCSMs) and passed off to the follicular B-cells

Question 34

What is the role of the follicular dendritic cells?

Answer 34

serve as antigen concentration sites for future selection and differentiation (used to test the antigen recognition and selection of memory B-cells during germinal centre reactions)

Question 35

What is the function of B-cell receptor clustering upon antigen binding?

Answer 35

Clustering of BCRs brings the tyrosine kinases in closer proximity to allow phosphorylation of the Igα/Igβ ITAM tyrosine residues

clustering facilitates internalisation of the antigen (BCR and antigen taken into cell, antigen processed and peptides presented to T-cell on MHC class II)

Question 36

Following stimulation of primary B cells at the T-cell/B-cell boarder within the lymph node, what may some B-cells proceed to do?

Answer 36

Some B-cells differentiate quickly into plasma cells that form primary foci and secrete an initial wave of IgM antibodies

Question 37

Other than forming primary foci and secreting an initial wave of IgM antibodies, what else might B-cells do following full T-dependent B-cell activation?

Answer 37

a few B-cells from the antigen-stimulated clones migrate from the extrafollicular foci to the primary follicles and form germinal centres
- within germinal centres, these B-cells proliferate rapidly (increases rate of mutation) to undergo somatic hypermutation in which the antigen recognition domain of the BCR is mutated
- affinity of mutated BCR for the antigen is tested using follicular dendritic cells (Affinity maturation)
- also site of class switch recombination (where the BCR switches out the constant domain to change the type of antibody produced dependent on the cytokine profile)

Question 38

What is the cytokine signal to promote class switching of B-cell from IgM to IgE?

Answer 38

Secretion of IL-4 from Type-2 T-helper cells.

Question 39

True or false: the antigen specificity of the class switched antibodies is the same?

Answer 39

True, only the Fc functional domain changes but the antigen antigen binding domain remains the same. - they all have different effector functions

Question 40

How does class switch recombination occurs?

Answer 40

Carried out by enzyme AID which deaminates cytidine residues resulting in double-strand breaks in switch regions upstream of the heavy chain constant region gene segments.
The DNA is recombined so that the constant μ region is excised and the VDJ region of the variable chain is now adjacent to a different constant region gene (such as gamma, delta, epsilon)

Question 41

Other than class switching recombination, what is another function of AID in T-dependent B-cell responses?

Answer 41

AID also mediates somatic hypermutation of B-cell receptors during germinal centre reactions by introducing double strand breaks which followed by DNA repair.

Question 42

What is the overall function of somatic hypermutation in the germinal centre reaction?

Answer 42

to produce B-cells with receptors that have increased affinity for the antigen in order to produce high-affinity plasma cells that secrete high affinity antibodies.

Question 43

Memory B-cells are produced during a ___(1)___ immune response from naive B-cells that do not differentiate into _______(2)_______

Answer 43

(1) primary
(2) plasma cells

Question 44

What is the purpose of memory B-cells?

Answer 44

reactivated by the specific antigen to yield a higher, faster, and stronger secondary humoral response

Question 45

What are long-lived plasma cells?

Answer 45

high affinity plasma cells arising from germinal centre reactions reside in the bone marrow (and other locations) and continually secrete antibodies to commonly encountered antigens that constantly circulate within the blood.

Question 46

How do TI-1 antigens interact with B-cells?

Answer 46

via both the BCR and innate immune receptors (PRRs)

Question 47

T-independent B-cell responses are normally generated by which types of B-cells and what do they give rise to?

Answer 47

B1 B-cells and MZ B-cells

Give rise to low affinity, primarily IgM antibodies (natural IgM antibodies)

Question 48

Describe the negative regulation of B-cell responses

Answer 48

• negative signalling through CD22 receptor on B-cells that contains an immunoreceptor Tyrosine-based inhibitory motif (ITIM)
• FcγRIIb receptor recognises the presence of IgG-containing immune complexes in the blood
• B-10 B-cells are a subpopulation of B-cells that release IL-10 upon antigenic stimulation, which has roles in reducing inflammation during an ongoing immune response and turns off immune responses