Mandl_T cell developement

Question 1

What antigen is a B cell receptor or antibody able to recognize?

Answer 1

• Soluble antigen in their native form
• Linear epitopes of sequential amino acid sequences
• Polysaccharide chains
• Lipids
• Secreted toxins

NOT Intracellular proteins (don’t access to the inside of the cell)
Can’t see what is intracellular —> so B cells miss a whole set of antigens —> that is when T cells come into play

Question 2

Which T cells binds to which MHC?

Answer 2

CD8 T cells → MHC-I
CD4 T cells → MHC-II

*CD4 and CD8 are the co-receptors to the TCR
TCR binds the peptide and the MHC and CD4/CD8 bind specifically to MHC of their prefered type

Question 3

What is the ratio of CD4+ : CD8+ T cells?
About how many different T cells are found in human and mice?

Answer 3

~ 2:1
CD4 : CD8

Human ~ 10^11 different T cells
Mice ~ 10^8 different T cells

Question 4

What is the structure of the T cell receptor?

Answer 4

2 TM glycoprotein chains that form a heterodimer → alpha and beta // delta and gamma

Extracellular portion of each chain consists of 2 domains → variable and constant
- No hinge regiond
- Connected by a disulfide bond
- TCR resembles the BCR Fab part, not the Fc (not there)
- TCR only has 1 Ag binding site (not 2 branches)
- Only exists in membrane bound form (no Ab secretion)
- Bind different antigens (intracellular for ex)
- Short TM domain and short cytoplasmic tail

Question 5

What is the T cell signal transduction complex like?
(Along with the TCR)

Answer 5

Signaling through the TCR requires the participation of a signal transduction complex: CD3

CD3 is formed by 5 invariant polypeptide chains that associate to form 3 dimers: ed, ge and zz (or zh) → each have ITAMs (zz has 3x ITAMs) in their intracellular portion

CD3 is required for membrane expression of TCR
- Include a total of 10 ITAM (Immunoreceptor Tyrosine-based Activation Motif) sequences
*Visualize the structure

*Signal transduction complex associated woth TCR, NOT the coreceptor (CD4/CD8)

Question 6

What are the structure of the T cell coreceptors?

Answer 6

TCR is non-covalently associated with accessory proteins that participate in signal transduction but only the co- receptors also recognize MHC

• CD4 is a monomeric membrane glycoprotein with 4 extracellular Ig domains (with disulfide bonds) → binds MHC-II (D1)
• CD8 is a disulfide-linked, ab heterodimer or aa homodimer → binds MHC-I

Question 7

What is the role of the T cell co-receptor in TCR signaling?

Answer 7

The co-receptor (CD4/CD8) binds to MHC → recruits LCK (T cell specific kinase) → LCK phosphorylates ITAMs on the TCR transduction complex (CD3) → Phosphorylated ITAMs recruit Zap70 (SH2 domains) → downstream signaling, Ca flux, cytoskeletal rearrangement, TFs, etc.

Question 8

What cellular changes does TCR-Antigen binding lead to?

Answer 8

• Metabolic changes
• Cytoskeletal changes
• Cell gets a lot bigger (protein production machinery)
• Ready for expansion

Question 9

What is an immunological synapse?

Answer 9

Antigen recognition → TCR-MCs formation → formation of an immunological synapse (cSMAC and pSMAC)

Immunological synapse = 3D interaction between the dendritical cell (of APC) and the TCR including many other proteins involved

cSMAC = central super-immunological center
pSMAC = peripheral super-immunological center

Question 10

How are the alpha and beta gene loci arranged? (TCR genes)

Answer 10

Both undergo somatic recombination

a-chain locus: LVx70-80 -…- Jx61 - Cx1

b-chain locus: LVx52 -…- Db1 - Jb1x6 - Cb1 - Db2 - Jb2x7 - Cb2

• Similar multigene organization to that of Ig genes
• Genes encoding the ab T cell receptor are only expressed in cells of the T cell lineage

Question 11

What process allows somatic rearrangement of TCR genes?

Answer 11

TCR gene segments are flanked by 12bp and 23bp spacer recombination signal sequences (RSS)
- Involves RAG1/2
- Excision circles are dissolved in division → can be used as intracellular indicators for recent thymic immigrants
*Called T cell receptor excision circles (TRECs)

*TdT is also present and adds diversity in T cells

Question 12

Give an example of virus that requires T cell recognition?

Answer 12

Listeria monocytogenes
Immune system needs to be able to detect intracellular antigens

Listeria takes advantage of the hosts actin and shoots itself between cells so its never visible to antibodies → T cells are critical to recognize it

Question 13

Why is the MHC so important to T cells?
What is it referred to in mice and humans?

Answer 13

In contrast to the Ig B cell receptor which can recognize an antigen alone, the T cell receptor only recognizes antigens that have been
processed and presented in the context of molecules encoded by the
MHC

Mice MHC = H-2 complex
Human MHC = HLA complex

Question 14

What are the different classes of MHC, their different gene regions and the product of those gene regions in humans?

Answer 14

MHC-I:
A, B and C regions of the gene → give respectively the following product: HLA-A, HLA-B, HLA-C
*1 MHC molecule/region
*same locus so inherited together

MHC-II:
DP, DQ, DR regions of the gene which respectively give the following gene products → DPαβ, DQαβ and DRαβ
*1 MHC molecule/region
*same locus so inherited together

MHC-III: (not antigen presenting)
C4, C2, BF regions which give the following products → C’ proteins and TNF-a, Lymphotoxins-a
*same locus so inherited together

The chain expressed determines the peptide that will be presented

Question 15

What are the different classes of MHC, their different gene regions and the product of those gene regions in mice?

Answer 15

MHC-I:
K region → H-2K MHC-I molecule
D region → H-2D or H2L MHC-I molecules
*not on the same locus?

MHC-II:
IA region → IAαβ MHC-II molecule
IE region → IEαβ MHC-II molecule
*same locus so inherited together

MHC-III:
S region → C’ proteins or TNF-α or Lymphotoxin-α (not Antigen presenting molecules)
*same locus so inherited together

Question 16

What does it mean for expression of MHC alleles to be codominant in 1 individual?

Answer 16

Both the mother and the father’s MHC alleles are expressed

An individual will express many different MHC molecules to be able to present a variety of different peptides

Also, a consequence of the codominance is that 2 unrelated individuals are highly unlikely to have exactly the same MHC haplotype
*MHC haplotype = set of MHC alleles

Question 17

What is the MHC haplotype of C57B6 mice?

Answer 17

*They all have the same haplotype because they are inbread so many times

Hapoltype b for all MHC alleles (H-2^b)

Can inject cells that have another haplotype into B6 mice as the mice immune cells won’t recognize the MHC

Question 18

What is the effect of crossing a mouse homologous for the b MHC locus with a mouse homologous for the k MHC locus (k haplotype)?

Answer 18

The progeny will be heterozygous → H-2^b/k

Since MHC alleles are codominant, the progeny will express all the following MHC-I molecules:
- H-2K^k
- H-2K^d
- H-2D^k
- H 2D^d
- H-2L^k
- H-2L^d

For MHC-II:
- IEα^k β^k
- IEα^k β^d
- IEα^d β^d
- IEα^d β^k
Etc.

Question 19

What is the difference between MHC-I and MHC-II in terms of the PEPTIDES they can present?

Answer 19

General MHC structure:
2 alpha helices on the sides between which the peptide is + 1 beta-sheet on the bottom on which the peptide sits

MHC-I peptides:
- 8-10 amino acids long because of the groves of the alpha helices
- anchor residues determined by MHC allele (P2, 3, 9) → determines which peptides will fit
*Peptide come from the inside of the cell

MHC-II peptides:
- 13-18 amino acids (longer as the alpha helices don’t bend into groves)
- lack conserved anchor residues
*Peptide come from the outside of the cells originally

Question 20

What is MHC restriction?

Answer 20

T cells have a dual specificity for self-MHC and for foreign antigen

• Need to recognize the MHC molecule + has to be specific to the peptide

*Known as MHC restriction because MHC molecule is said to restrict the ability of T cells to recognize Ag

Question 21

What experiment was done to demonstrate MHC restriction?

Answer 21

1. Injected LCMV into H-2^k mice
2. Plated the splenic T cells into 3 dish (these T cells were antigen specific for LCMV because the mouse had been immunized):
   - H-2^k target cells → no recognition of the peptide (no LCMV)
   - H-2^k LCMV-infected target cells → recognition of the MHC and the peptide
   - H-2^b LCMV-infected target cells → no matching of MHC

*Measured T cell activity using chromeome release as a mean of cytotoxicity

Question 22

What experiment confirmed the MHC restriction/Specific duality of T cells?

Answer 22

Crystal structure of TCR-pMHC interaction

Question 23

Where does the CD4 and the CD8 bind to the MHC?

Answer 23

• The binding site for CD4 on the MHC molecules class II lies at the base of the β2 domain of an MHC class II molecule between the β2 and a2 domains (base of MHC-II)
• The binding site for CD8 on the MHC-I involves the base of the binding a1and a2 and weakly bound to a3 domain (a1and a2 are the peptide binding portion and a3 is at the base)


Question 24

Airway epithelial cells infected with coronavirus will:
a) Present peptides on MHC-II and be recognized by CD8 T cells
b) Be engulfed by macrophages who will present peptides on MHC-II and be recognized by CD4 T cells
c) Present peptides on MHC-I and be recognized by CD8 T cells

Answer 24

b) Be engulfed by macrophages who will present peptides on MHC-II and be recognized by CD4 T cells
c) Present peptides on MHC-I and be recognized by CD8 T cells

A is not true because CD8 T cells don’t recognize MHC-II, they recognize MHC-I

Question 25

What is early T cell lineage dependent on (at lymphoid precursor stage)?

Answer 25

Lymphoid precursors migrate from the bone marrow to the thymus via the blood: commitment to T cell lineage dependent on NOTCH

Question 26

How does thymic function change with age?

Answer 26

It declines with age. Young thymi are large et generate lots of immature T cells As you get older, the thymus shrinks and adults rely a lot on T cells that were made at earlier age

Question 27

At what stages of T cell development does somatic rearrangement occur?

Answer 27

1. Beta chain rearranges in DN3-DN4 stages - D - J recombinations - V - DJ recombination 2. Test b-chain through the pre-TCR (to allow surface expression, need a surrogate alpha chain) 3. Alpha chain rearranges in DP stage - V - J recombination Total T cell development in mice ~ 3 weeks

Question 28

What markers differentiate the DN stages?

Answer 28

CD44 and CD25 DN1 = CD44+ CD25- DN2 = CD44+ CD25+ DN3 = CD44- CD25+ DN4 - CD44- CD25-

Question 29

At what stages does somatic recombinatin occur for gamma delta TCR chains?

Answer 29

Both at the DN2, DN3 stage

Question 30

How is T cell development spatially and temporally organized in the thymus?

Answer 30

Distinct phases of development occur in distinct microenvironments – thymic cortex: immature thymocytes – thymic medulla: more mature thymocytes 1. Enter as thymic precursors from the blood (coming from BM) → medulla (DN1) 2. Migrate to the cortex and become DN2 → DN3 → DN4 → DP → positive selection 3. Migrate back to the medulla as DP → SP + undergo negative selection 4. Leave the medulla → blood as mature SP 5. Egress from thymus requires expression of sphingosine-1-phospate receptor S1PR1 (strong chemotaxic signal which is high in the blood and low in the tissues) 6. RTE (recent thymic emigrants) join the mature peripheral T cell pool of naïve T cells

Question 31

What are key point of positive selection?

Answer 31

T cells which express an ab TCR that recognize self- MHC:self-peptide complexes are allowed to mature TCR recognizes MHCII → positively selected to CD4+ lineage TCR recognizes MHCI → positively selected to CD8+ lineage *Requires interation with cortical TECs The process of positive selection enforces MHC restriction + selects T cells that are most "effective" T cells that are not positively selected dye by neglect *This process implies that all T cells in our body have some level of self-reactivity

Question 32

How does positive selection enforce MHC restriction?

Answer 32

Only TCRs that interact with (i.e. “recognize”) self-MHC will enter the periphery - Moderate affinity for self-MHC/self-peptide (ex: thymic epithelium expresses MHCa, then you TCRs have to recognize it) T cells are seeing specifically the MHC alleles you have in your body → Ensures matching between random generation TCR and the MHC alleles present self-expressed

Question 33

What experiment allows to assess which thymic compartment requires MHC expression for normal positive selection?

Answer 33

Bone Marrow chimeras: MHC-II KO and WT mice are used * cTECs are radiation resistant while hematopoietic cells are not WT → MHC II KO: - No CD8+ T cells in the thymus nor in the Lymph Nodes (can't pass positive selection because don't recognize MHC-II on cTECs) MHC II KO → WT - Normal CD8+ T cell development

Question 34

What are important features of Negative Selection?

Answer 34

Deletion of ab T cells which bind too strongly to self- peptide:MHC = Clonal deletion Interaction with medulla thymic epithelial cells (mTECs), dendritic cells, thymic B cells (professional APCs)

Question 35

What does the affinity model of T-cell selection show?

Answer 35

TCR affinity for self-peptide:self-MHC follows a bell-curve distribution Low affinity → death by neglect (fail positive selection) Mid-high → Selected Mid-high+ → agonist selection of Tregs High → Clonal deletion (fail negative selection)

Question 36

What is T cell precursor frequency? How can is be tested experimentally?

Answer 36

Every cell has a unique TCR, but they can have specificity for the same MHC:peptide A "T cell precursor frequency" refers to the proportion or number of naive T cells within the immune system that are capable of recognizing a specific antigen More diversity in the repertoire/lower precursor frequency → higher probability to have a TCR that recognizes the peptide:MHC, but slower Less diversity/higher precursor frequency → lower probability of recognizing a specific antigen, but faster response when recognized Can test experimentally with tetramers → allows to detect T cells specific for a given antigen (peptide:MHC) - CD8+ T cells have a higher precursor frequency (more antigen specific CD8 T cells for a given antigen)

Question 37

What is a Naïve T cell? How does it circulate in the body?

Answer 37

Naïve T cells: mature T cells in the periphery which have not encountered their specific antigens Constant recirculation: Blood → Lymph node(/Spleen) (~12h) where they encounter different APCs and sample the peptides → efferent lymph → blood *Only activated T cells can enter non-lymphoid tissues due to expression of homing receptors

Question 38

How do naïve T cells enter the lymph nodes?

Answer 38

They enter through high endothelial venules (HEV) → specialized vascular endothelial cells expressing receptors that allow T cell crossing from blood to tissue 1. Rolling - All naïve T cells express CD62L → binds to PNAd on endothelial cells - Rolling allows integrin activation 2. Activation - CCR7:CCL19/21 - CCL19/21 = chemokine expressed in the lymph nodes 3. Adhesion LFA-1:ICAM-1/2 VLA-4:VCAM1/fibronectin 4. Transmigration

Question 39

How do lymphocytes exit the lymph nodes?

Answer 39

Lymphocyte exit is through lymphatic sinuses in cortex or medulla Egress requires expression of S1PR1 by lymphocytes

Question 40

What process leads to antigen encounter events? What are 2 places antigen capture can be done?

Answer 40

1. Infection in tissue causes free antigen and antigen-bearing dendritic cells (tissue-resident) to enter lymph and travel to draining lymph node - Antigen can also arrive via the lymph (lymph-resident DC sampling the lymph), is captured by dendritic cells and then presented to T cells (fast) - Antigen can be captured at the site of infection by dendritic cells and the dendritic cells migrate to the LN in a CCR7-dependent manner (slower) 2. Naïve T cells enter dLN through HEV, encounter antigen, are activated and expand (long interaction when it recognizes the peptide:MHC) 3. Following clonal expansion, effector T cells leave dLN through lymphatics, re-enter blood and home to infected tissue site where they clear infection *Before activation, they can't enter these tissues

Question 41

Does T cell activation lead to in the lymph nodes?

Answer 41

T cell activation transiently leads to down-regulation of S1PR1 → can't leave the lymph node - Effector differentiation, clonal expansion and activation is done in the lymph node - Then, S1PR1 is re-upregulated to allow effector T cells to go fight the infection at its site *Only activated T cells can enter non-lymphoid tissues due to expression of homing receptors

Question 42

What does priming of naïve T cells require?

Answer 42

Priming = the activation and differentiation of naïve T cells upon cognate antigen encounter APCs deliver 3 signals to naïve T cells: 1) TCR-pMHC interaction → activation 2) Co-stimulatory signal such as B7 molecule on APC interact with CD28 on T cells → leads to IL2 expression → essential survival signal 3) Cytokines secreted by APC needed for effector T cell differentiation

Question 43

What is IL-2 and its subparts?

Answer 43

IL-2a = CD25 IL-2b = CD122 Naive T cell expresses y and b chains → moderate affintity to IL-2 Activated T cell → expression of the alpha chain which completes the IL-2 receptor → high affinity binding to IL-2 *Strong survival signal, required for T cells to divide

Question 44

What is the specific role of CD8+ effector T cells? What do they require for priming? What cytokines do activated CD8+ T cells secrete?

Answer 44

Important in defense against INTRACELLULAR pathogens, especially viruses, & tumor cells Most CD8 T cell responses require CD4 T cell help during priming → CD4 helper T cells interacts with APC which leads to secretion of adequate cytokines for CD8 T cell activation CD8 T cells are serial killers of target cells that express antigen Can also act by producing cytokines: IFNy and TNFa

Question 45

How to CD8+ effector T cells kill their target cells?

Answer 45

Only activated CD8+ effector T cells can kill target cells upon pMHC recognition - All cells are susceptible to killing by armed CD8 TL but killing requires specific recognition by TCR - CD8+ T cells kill their targets by inducing apoptosis by release of cytotoxic granules containing perforin and granzymes *Very local event, require close contact Perforin → perforates forms pores in the target cell plasma membrane Granzyme → activate caspase and damages the mitochondria, enters the target cell by the pores *1 signal for killing / 3 signals for activation

Question 46

What are different subsets of CD4+ effector T cells and their functions?

Answer 46

TH1 cells → virus, intracellular pathogens, promote class-switching of Abs TH2 cells → Allergy response, Extracellular pathogens (Helminth, parasites), recruitment of eosinophils, basophils, mast cells TH17 cells → recruitment of neutrophils, Extracellular bacteria, fungi T FH cells → Help B cells become activated for Ab production Treg cells → immunomodulatory role, lack T cell activation

Question 47

What are 3 properties that distinguish Helper CD4+ T cells subsets? Give examples for each.

Answer 47

1. Polarizing cytokines (different cytokines lead to differentiation into specific subsets) 2. Master gene regulator 3. Signature set of effector cytokines Ex: IFN-y/IL-12 → T-bet expression (TF) → TH1 differentiation → IFNy secretion Ex: IL-4 → GATA-3 expression (TF) → TH2 differentiation → IL-4/IL-5/IL-13 secretion Ex: IL-6/IL-23/TGF-b → RORyT expression (TF) → TH17 differentiation → IL-17/IL-22 secretion Ex: IL-6 → Bcl-6 expression (TF) → T FH differentiation → IL-21 secretion Ex: TGF-b/IL-2 → FoxP3 expression (TF) → Treg differentiation → TGF-b/IL-10 secretion