Mandl_T cell developement Flashcards
What antigen is a B cell receptor or antibody able to recognize?
- 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
Which T cells binds to which MHC?
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
What is the ratio of CD4+ : CD8+ T cells?
About how many different T cells are found in human and mice?
~ 2:1
CD4 : CD8
Human ~ 10^11 different T cells
Mice ~ 10^8 different T cells
What is the structure of the T cell receptor?
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
What is the T cell signal transduction complex like?
(Along with the TCR)
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)
What are the structure of the T cell coreceptors?
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
What is the role of the T cell co-receptor in TCR signaling?
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.
What cellular changes does TCR-Antigen binding lead to?
- Metabolic changes
- Cytoskeletal changes
- Cell gets a lot bigger (protein production machinery)
- Ready for expansion
What is an immunological synapse?
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
How are the alpha and beta gene loci arranged? (TCR genes)
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
What process allows somatic rearrangement of TCR genes?
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
Give an example of virus that requires T cell recognition?
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
Why is the MHC so important to T cells?
What is it referred to in mice and humans?
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
What are the different classes of MHC, their different gene regions and the product of those gene regions in humans?
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
What are the different classes of MHC, their different gene regions and the product of those gene regions in mice?
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
What does it mean for expression of MHC alleles to be codominant in 1 individual?
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
What is the MHC haplotype of C57B6 mice?
*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
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)?
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.
What is the difference between MHC-I and MHC-II in terms of the PEPTIDES they can present?
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
What is MHC restriction?
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
What experiment was done to demonstrate MHC restriction?
- Injected LCMV into H-2^k mice
- 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
- H-2^b 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
What experiment confirmed the MHC restriction/Specific duality of T cells?
Crystal structure of TCR-pMHC interaction
Where does the CD4 and the CD8 bind to the MHC?
- 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)
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
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
What is early T cell lineage dependent on?
Lymphoid precursors migrate from the bone marrow to the thymus via the blood: commitment to T cell lineage dependent on NOTCH
How does thymic function change with age?
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
At what stages of T cell development does somatic rearrangement occur?
- Beta chain rearranges in DN3-DN4 stages
- D - J recombinations
- V - DJ recombination - Test b-chain through the pre-TCR (to allow surface expression, need a surrogate alpha chain)
- Alpha chain rearranges in DP stage
- V - J recombination
Total T cell development in mice ~ 3 weeks
What markers differentiate the DN stages?
CD44 and CD25
DN1 = CD44+ CD25-
DN2 = CD44+ CD25+
DN3 = CD44- CD25+
DN4 - CD44- CD25-
At what stages does somatic recombinatin occur for gamma delta TCR chains?
Both at the DN2, DN3 stage
How is T cell development spatially and temporally organized in the thymus?
Distinct phases of development occur in distinct microenvironments
– thymic cortex: immature thymocytes
– thymic medulla: more mature thymocytes
- Enter as thymic precursors from the blood (coming from BM) → medulla (DN1)
- Migrate to the cortex and become DN2 → DN3 → DN4 → DP → positive selection
- Migrate back to the medulla as DP → SP + undergo negative selection
- Leave the medulla → blood as mature SP
- 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)
- RTE (recent thymic emigrants) join the mature peripheral T cell pool of naïve T cells
What are key point of positive selection?
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
How does positive selection enforce MHC restriction?
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
What experiment allows to assess which thymic compartment requires MHC expression for normal positive selection?
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
What are important features of Negative Selection?
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)
What does the affinity model of T-cell selection show?
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)
What is T cell precursor frequency?
How can is be tested experimentally?
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)
What is a Naïve T cell?
How does it circulate in the body?
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
How do naïve T cells enter the lymph nodes?
They enter through high endothelial venules (HEV) → specialized vascular endothelial cells expressing receptors that allow T cell crossing from blood to tissue
- Rolling
- All naïve T cells express CD62L → binds to PNAd on endothelial cells
- Rolling allows integrin activation - Activation
- CCR7:CCL19/21
- CCL19/21 = chemokine expressed in the lymph nodes - Adhesion
LFA-1:ICAM-1/2
VLA-4:VCAM1/fibronectin - Transmigration
How do lymphocytes exit the lymph nodes?
Lymphocyte exit is through lymphatic sinuses in cortex or medulla
Egress requires expression of S1PR1 by lymphocytes
What process leads to antigen encounter events?
What are 2 places antigen capture can be done?
- 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)
- Naïve T cells enter dLN through HEV, encounter antigen, are activated and expand (long interaction when it recognizes the peptide:MHC)
- 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
Does T cell activation lead to in the lymph nodes?
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
What does priming of naïve T cells require?
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
What is IL-2 and its subparts?
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
What is the specific role of CD8+ effector T cells?
What do they require for priming?
What cytokines do activated CD8+ T cells secrete?
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
How to CD8+ effector T cells kill their target cells?
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
What are different subsets of CD4+ effector T cells and their functions?
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
What are 3 properties that distinguish Helper CD4+ T cells subsets?
Give examples for each.
- Polarizing cytokines (different cytokines lead to differentiation into specific subsets)
- Master gene regulator
- 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
