T cell development, activation and differentiation Flashcards
In which organ does the T cells develop?
T cells originate from HCS that develop into common lymphocyte progenitors in the bone marrow, and then migrate to the thymus before even committing to the T cell lineage. If they do commit to the T cell lineage, they go through several developmental stages and selection in the thymus and enter the periphery as mature naive T cells. So T cell development occurs in the thymus.
How does the development of T cells change over time?
Thymocytes are produced throughout our lifetime, although after puberty the thymus shrinks (involutes) and produces fewer and fewer T cells over time. You are able to live without a thymus if it’s removed in adulthood, but not if you’re prepubescent, especially very young children that require a thymectomy get’s extremely susceptible to infections.
What commits a precursor cell to the T cell lineage?
The expression of the Notch receptor and subsequent Notch signalling. Cells that have started to mature into T cells are still multipotent until this receptor is expressed, in the DN2 stage.
Go through the early stages of T cell development step-by-step.
T cell precursors enter the thymus via blood vessels in the corticomedullary boundary (between medulla and cortex). At this stage, the thymus-settling progenitors (TSPs) are double negative (DN1). The TSPs encounter Notch ligands as soon as they enter the thymic environment and start to gradually commit to the T cell lineage and travel to the cortex.
In the cortex, they start to proliferate and start to express surface CD25 and now they have reached the DN2 stage. At this stage, early, the genes for the TCR γ, δ, and β chains start to rearrange (recall that most T cells have αβ) but the TCR α chain locus is still inaccessible. in the late DN2 stage, T cells fully commit to the T cell lineage.
In the transition from DN2-DN3 the cells continue to rearrange the TCR γ, δ, and β chains and here the first decision is made, if the cell should join the TCR-γδ or TCR-αβ T-cell lineage. If the cell successfully rearrange the join their β chain, they typically commit to the TCR-αβ T-cell lineage and prepare to enter the DN4 stage. If the cell successfully rearranges the γ and δ chains instead (which is typically slower as two successful independent rearrangements need to happen, the reason to why TCR-αβ T cells are more common) they leave the thymus as mature DN (CD4 CD8 ) T cells which recognize unconventional antigens.
Cells in the transition between the DN3 and DN4 stage have successfully rearranged their TCR β chains, and go through a process called β-selection, in which an invariant pre-Tα chain is expressed that act as a surrogate which assembles with a successfully rearranged and translated β chain, as well as CD3 complex proteins to form a pre-TCR. If it can successfully assemble, the pre-TCR signalling induces rapid proliferation (and suppression after a few rounds) and suppression of further rearrangement of the β chain, allelic exclusion and initiation of α chain rearrangement. This leads to maturation into the DN4 stage.
During the DN4 stage, rearrangement of the α chain occur and once a successfully rearranged α chain replace the surrogate α chain the complete αβ TCR has formed, and the cells start to express both CD4 and CD8, and is ready for the second stage of T-cell development: selection.
Describe the T cell selection process in detail.
First, the DP T cells undergo positive selection in the cortex, where only those that are able to bind to MHC molecules are selected, those T cells that bind class II MHC (with their CD4 molecule) loose expression of CD8 and become CD4+ single positive. Those that bind to MHC I become CD8+ SP. All the positively selected cells here go on in the selection process, the rest die from anergy (from not binding MHC - not getting survival signals) and the majority of thymocytes die here (~95%) aka death by neglect. This process is referred to as MHC restriction.
Then the now SP cells go through negative selection (mostly in the medulla), where they are tested for autoreactivity mediated by mTECs/AIRE. Those that bind with too high affinity to self-MHC/self-peptide complexes are selected against and die by apoptosis. Those which bind with low/intermediate affinity are selected for. So they still have some affinity to self, but not enough to elicit a response and this provides self-tolerance (central tolerance).
Cells that have been selected for all the way mature into Naive T cells that go into the periphery (only 2% of the total amount of DP cells get here! a lot of investment to make sure the T cells are thoroughly screened).
Note: It’s not certain that these selection processes occur sequentially, most likely negative selection can occur at various points in development.
Describe thymic selection in short.
- Positive selection: Selects thymocytes bearing receptors capable of binding self-MHC molecules with low- intermediate affinity, resulting in MHC restriction.
- Negative selection: Selects against thymocytes bearing high-affinity receptors for self-MHC/peptide complexes,
resulting in self-tolerance.
Note: some T cells with high affinity to self antigens will develop into Tregs.
How are the T cells tested against tissue specific proteins from all over the body when these cell types are not present in the thymus?
T cells are tested for autoreactivity by medullary thymic epithelial cells, which express a unique protein called AIRE, short for “autoimmune regulator. The mechanism of action of AIRE is that it binds to epigenetic marks on histones associated with closed chromatin and recruit transcription factors to these silenced promoters, allowing RNA polymerase to gain access and transcribe the proteins. The proteins are then presented on the membrane to screen the SP cells for autoreactivity in a safe and controlled way before maturation.
Other thymic stromal cells, including dendritic cells and B cells also present self-proteins involved in antigen presentation and B-cell function and are able to induce apoptosis in the SP T cells if they are autoreactive.
DP thymocytes may commit to other lineages, which can they commit to instead of classical CD8+/CD4+ T cells?
DP thymocytes can commit to become:
- NKT cells: play a role in innate immunity and share features with both T and NK cells. They express a TCR with an invariant TCRα chain (Vα14). NKT cells Interact with CD1 molecules on APCs presenting lipid antigens.
- Intraepithelial lymphocytes (IELs): Usually CD8+, but also have features of innate immune cells. IELs partol barrier tissues.
- Regulatory T-cells (Tregs): a CD4+ subset that helps to quench adaptive immunity
All three of these cell types can develop from DP thymocytes in response to autoreactive, high-affinity TCR interactions—the same interactions, in fact, that mediate negative selection. The signaling cues for this alternative development are unclear at present.
How long does it take for DP thymocytes to become SP and how long does it take for a SP thymocyte to leave the thymus?
It takes less than 3 days for a newly formed DP thymocyte to mature to the SP stage, if positively selected. SP cells spend a longer period (from 4 to 12 days) in the medulla, browsing the surface of epithelial and dendritic cells for self antigen before being given permission to leave the thymus.
What is needed for a mature thymocyte to leave the thymus?
To leave the thymus, the cell need to receive positive-selecting TCR signals which upregulates the transcription factor Foxo1.
- Foxo1 upregulates the expression of Klf2 which in turn upregulates sphingosine 1-phosphate receptor 1 (S1P-R). The exit of SP thymocytes from the thymus depends on expression of the and its interaction with S1P at the corticomedullary junction.
- Foxo1 also upregulates IL-7R (giving survival signals) and CCR7 (a chemokine receptor that helps cells exit and move to lymph nodes).
What are newly mature cells that exit the thymus referred to as? Describe them briefly.
Newly mature cells that exit the thymus are referred to as as recent thymic emigrants (RTEs). RTEs can be distinguished from the majority of peripheral naive T cells because their levels of expression of several surface proteins (they are more like their immature ancestors than their naive T cell descendants). They are not yet optimally functional and undergo a post-thymic phase of maturation in secondary lymphoid tissue that fully licenses them as naive T cells.
What is the role of Tregs? What characterizes them?
Tregs negatively regulate immune responses, mainly by browsing for autoreactive T cells that have managed to escape the negative selection in the thymus.
Tregs belong to a subset of CD4+ T-cells that characterized by the expression of FoxP3 transcription factor.
Negative selection in the thymus is not perfect, thankfully we have peripheral tolerance mechanisms to handle it. What could cause autoreactive T cells to escape the thymic selection?
Autoreactive T cells do escape, either because they have too low an affinity for self to induce clonal deletion, or because they happen not to have browsed the “right” tissue-specific antigen/MHC combination.
What are the four mechanisms by which Tregs regulate immune responses?
Tregs have four ways of negatively regulating the immune response:
- They can deplete the local area of stimulating cytokines: by expressing high affinity IL-2 receptors and can compete for the cytokines that activated T cells need to survive and proliferate.
- Produce inhibiting cytokines: including IL-10 and TGF-β, which bind receptors on activated T cells and reduce signaling activity.
- Inhibit APC activity: Tregs can interact directly with MHC class II–expressing APCs and inhibit their maturation, leaving them less able to activate T cells.
- Directly kill T-cells (cytotoxicity): Tregs can also display
cytotoxic function and kill cells by secreting perforin and granzyme.
Page 639 in the book.
Other than Tregs, name another mechanism that contribute to peripheral tolerance.
Regulatory T cells contribute to peripheral T-cell tolerance, as does the strict requirement for costimulatory interactions in order to activate a T cell. Costimulation can be provided only by professional antigen-presenting cells, whose activity is highly regulated - which further limits T cell activation.
(Some self-antigens are presented by non-APCs, preventing initiation of autoimmunity. Strong self-antigen signaling through the TCR in the absence of costimulation may drive the T-cells into anergy)
Briefly describe the events that activate the adaptive immune response.
Antigen presentation is the first signal needed for T cell activation. Upon infection, the innate immune system gets alerted to the infection and APCs gets activated via PRR signalling. The APCs have either opsonized (engulfed) the pathogen or are infected by intracellular pathogens, and have processed and presented peptides from the pathogen on MHC class I or II molecules and migrated to secondary lymphoid tissues, including the lymph nodes and settled in T cell zones. Here they are scanned by naive CD4+ and CD8+ T cells for a possible match. If a match is made, the adaptive immunity gets activated.
Depending on the antigen that activate the APCs, they will be able to “tune” the T cell response.
Describe the phenotype of naive T cells.
Naive T cells have not yet encountered antigen. Their chromatin is condensed, they have very little cytoplasm, and they exhibit little transcriptional activity. However, they are mobile cells and recirculate continually among the blood, lymph, and secondary lymphoid tissues, including the lymph nodes, browsing for antigen.
What would explain why naive T cells recirculate so much?
Since the diversity of antigen recognition is so high, only about 1/10^5 is likely to be specific to a specific antigen. It is estimated that each naïve T cell recirculates from blood through lymph nodes and back again every 12 to 24
hours to increase the chances of finding “its” antigen. If it still don’t find any, it can enter various tissues and out again until it finds a match.
What three signals are needed for T cell activation?
Signal 1: antigen-specific TCR engagement (TCR/CD3-peptide/MHC interaction)
Signal 2: costimulatory receptor contact with costimulatory ligands (Such as the CD28 receptor on T cell making contact with it’s ligands CD80 and CD86 expressed by pAPCs)
These two signals make up the “two-signal hypothesis” which was initially found to be needed for T cell activation, but now we know a third signal is needed for full T cell activation:
Signal 3: cytokines directing T- cell differentiation into distinct effector cell types (soluble or polarizing cytokines).
What happens upon T cell activation by APCs?
Upon APC mediated activation (and the rest of the necessary signals) the T cells differentiate into their effector forms:
- CD8+ T cells can become cytotoxic T cells or memory cells. T cells that kill infected “target cells” and are capable of macrophage activation.
- CD4+ T cells become helper T cells or memory cells: Cells that activate B cells, macrophages and other cells.
Describe T cell activation in detail, including the cSMAC and pSMAC.
A successful T cell–APC interaction results in the stable organization of signaling molecules into an immune synapse. The TCR/MHC-peptide (+ CD4/CD8 receptors) complexes aggregate in the middle and the costimulatory interactions stabilize the cell-cell interaction (together higher avidity). All this forms the central supramolecular activating complex, or cSMAC, the central part of immunological synapses. Adhesion molecules/bound ligands peripherally localize and form the peripheral supramolecular activating complex, pSMAC.
Describe the initiation of TCR signalling.
The CD4 and CD8 coreceptors help to stabilize the TCR/MHC-peptide interaction, but also have another key function: They associate with the tyrosine kinase Lck. The TCR is associated with the CD3 complex, which have several ITAMs on their cytoplasmic domains. Upon TCR activation, the Lck phosphorylates the ITAMs on the CD3 complex, which induces a conformational change allowing ZAP-70 to dock and be phosphorylated and activated by Lck –> starting a cascade that leads to activation of genes that regulate survival, proliferation and differentiation into effector cells.
Name and describe one costimulatory T cell receptor.
CD28 is a 44 kDa glycoprotein homodimer expressed by the majority of T cells, that binds to the ligands CD80 and CD86 expressed by pAPCs (also medullary thymic epithelial cells). CD28 functions in activation of naïve T cells. CD28 signalling enhances TCR-induced proliferation and survival by cooperating with T-cell receptor signals to induce expression of the pro-proliferative cytokine IL-2 and the prosurvival Bcl-2 family member, Bcl-x.
ICOS (inducible costimulator) is expressed by effector and memory T cells and binds to ICOS-L expressed by B cells, some APCs, and T cells and mediates maintenance of activity of differentiated T cells; a feature of T-/B-cell interactions.
How does the expression of CD80/86 differ among the pAPCs?
Mature DCs constitutively express CD80/86, and macrophages and B cells have the capacity to up-regulate CD80/86 after they are activated by an encounter with pathogen.
