Immunology Lesson 10: T and B Cell Maturation (PDF)

Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 T&B CELL MATURATION Both B and T cells derive from hematopoietic stem cells that will develop into their common lymphoid progenitor; its migration into the thymus will give rise to T cells, meanwhile its migration into the bone marrow will give rise to B cells. This tissue-specific differentiation is possible thanks to membrane receptor signals that regulate specific gene transcription. The common progenitor differentiates into B cells, after the opening of its chromatin, thanks to the action of EBF, E2A and PAX5 transcriptional factors that will lead to the BCR rearrangement. Ultimately the B cell formed will differentiate into follicular B cells (in the periphery), marginal zone B cells or B1 cells. For the T cell differentiation, the transcription factor involved are Notch1 and GATA3. In this case, after the opening of the chromatin, there will be the TCR rearrangement. COMMON STEPS 1. The early steps in their maturation depend strongly on cytokines that will lead to a first wave of proliferation. The cytokines involved are IL-4 for the B cells and IL-7 for the T cells. Mutations in the γ common chain of the cytokine receptor can lead to the X-linked Severe Combined Immunodeficiency Disease (X-SCID) in which patients possess no B and T cells. The gene responsible for the γ common chain resides on the X Chromosome. 2. Rearrangement of genes coding for the antigen receptor that are crucial in signalling for cell survival, proliferation and continuation in differentiation. Mutations or defects that occur at this stage can prevent normal cell development One example is the Bruton Disease in which B cells lack the Bruton-tyrosine kinase and thus they will not be able to mature and work properly in case of infections. 1 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 3. If the antigen receptor formed can’t produce a correct signalling, the cell will receive a stop signal that will block the second wave of proliferation. If the antigen receptor function properly, the cell won’t receive the stop signal and there will be a second wave of proliferation. This regulation is possible thanks to two checkpoints. 4. Finally, positive and negative selection will lead to the final functional lymphocytes. This selection is mediated by different mechanism and is crucial in preventing autoimmune diseases by eliminating cells that recognize self-molecules and attack the host own cells. THYMUS Located in the thorax, posterior to the sternum Composed of a capsule and many trabeculae o that act as supporting structure Each lobe is subdivided into more lobes Site of T cell development Most active during the childhood and its activity decreases from adolescence to adulthood DiGeorge Syndrome Affected patient possesses a non-functional thymus and thus their T-cell can’t undergo maturation. Memory B-cells are also affected because their differentiation is dependant to T-cell activity Pathology characterized with many infections to which patients recover poorly and patients which possess no mature T cells and no memory B cells, but only the IgM isotype Being the T cells, CD3+, it’s possible to make the T lymphocytes clearly visible by Staining the thymus with a proliferation Marker using antibodies that target the CD3 (Anti Ki67Antibodies) active proliferating cells molecule will be stained in brown. 2 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 In the thymus are also present: o Cortical Epithelial cells o Macrophages and Dendritic cells o Newly arrived T-lymphocytes, driven here by the action of NOTCH signalling when they were still pro-T lymphocytes Thymocytes are intimately associated with the epithelial cell as they develop into the thymus. In this picture are also very clear the holes that the thymocytes leave into the epithelial by their association T-CELL MATURATION Immature cells arriving at the thymus do not yet express the antigen receptor (TCR) nor the CD3 transducer chains: they are CD3- cells. Signals from thymic epithelial-stromal cells induce the generation of TCR through the complex gene recombination. In this way, each T cell randomly acquires a different TCR able to react with a distinct ligand. Thanks to this phenomenon, we are prepared to react to almost any pathogens that we can encounter in our life. The first chain that undergoes arrangement is either the β or the γδ. The correct rearrangement of the first chain is also the first checkpoint for the correct formation of the T-cells. If the T-cells fail to rearrange the chain, it undergoes apoptosis If the β chain (or γδ) is correctly rearranged, it’s functionality (if it works) must also be checked. This is done thanks to a surrogate chain termed Ψα (ti-alpha) monomorphic chain. If the β chain (in the cytosol) can bind the Ψα chain (also in the cytosol), the resulting dimer will be expressed on the cell surface. 3 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 The Ψα-β dimer, then, interact with the thymic cells. If the surrogate TCR transduces a signal it means that the Β chain is working well. The resulting signal will then: 1) block further VDJ rearrangement of the β chain sequence and interrupts also the VDJ rearrangement of TCRG loci (allele exclusion); 2) induce the lymphocyte proliferation that will lead to many lymphocytes having the same β-chain that can start to rearrange the α chain; Double positive stage: Once immature T cells express their individual pre-TCR, they become CD3+ and express both CD4 and CD8 proteins (CD3+CD4+CD8+). T cells of this differentiation stage are called double positive cells because possess both coreceptors. Death of the neglected: abbandona Double positive thymocyte express now a TCR able to bind HLA-peptide (HLA-p) on the surface of thymic epithelial stromal cells. This binding will result in an anti-apoptotic signal delivered by the TCR-CD3 chains. Thymocytes that can’t bind the HLA-p won’t receive this signal and will undergo apoptosis. Negative selection: Double positive thymocytes expressing a TCR that binds HLA-p with too much strength also need to be eliminated and thus undergo clonal deletion. This is done in order to avoid the presence of thymocyte that react against self-peptides which is very common due to the random rearrangement of the TCR. Negative selection is, thus, a dramatic event since most of the T cells die (80%). A few of them, however, may re-make the TCR or become natural regulatory T cells. Treg cells are also crucial to this passage because can suppress the reactions against self. This negative selection of the TCR is driven in the thymic medulla more efficiently by bone-marrow-derived-antigen-presenting-cells than by thymic cortical and medullary epithelial cells. Single positive stage: Ultimately, T cells need to possess only one of the two coreceptor. This is decided by the first coreceptor that bind the relative HLA-peptide. T cells with a TCR binding first Class I HLA-p become single positive CD3+CD8+ cells (A); T cells with a TCR binding first Class II HLA-p become single positive CD3+CD4+ cells (B); The coreceptor that won’t bind will be discarded. In the image: at the beginning, both the co-receptors are present. Then, in the image A the cell interacts with the HLA class II, so only the CD4+ co-receptor remains expressed, and the same thing happens with HLA class I and CD8+ receptors in the image B. A) B) 4 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 In the Subcapsular region resides immature double-negative thymocytes that undergo positive selection in the cortex thanks to strict contact with the cortical epithelial cells resulting in immature double positive thymocytes. These thymocytes then undergo negative selection in the medulla with the help of Dendritic cells (more efficient) and Epithelial cells. Ultimately, mature CD8+ or CD4+ T cells are produced. T-cells that exit from the thymus are only the ones that have a TCR able to bind the Histocompatibility molecules, containing a self-peptide, at Low affinity. o High affinity → Risk of self-harm o No Affinity → The TCR may not work at all B CELLS MATURATION The virgin mature B cell emerging from the bone marrow is histologically similar to a naïve T cell o Both possess a big nucleus with few cytoplasm around it. Cells committed to the B cell lineage in the bone marrow express cell surface glycoproteins CD45 (B220) and CD19. o Used as markers in the laboratory Daily we produce 5 x 107 new B cells but only 5 x 106 of them (10%) survive because of mistakes, competition and negative selection. The first step of the B cell maturation is an early proliferation in response to cytokines. This is done in order to obtain a pool of many cells that will make a wide B cell repertoire. It starts from the stem cell possessing the cytokine receptor for IL-3 and CD19 that will be maintained during the whole B cell development Another important component that the B cell must have, is the one responsible for interaction with adhesion molecules. One example is the CD44 that interact with Hyaluronic Acid. 5 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 If the binding of IL-3 and the Adhesion molecule has a positive outcome, the stem cell will receive a proliferation signal and will maturate into a pro-B cell. The maturation into a pro-B cell can be divided into an early phase and a late phase and these different phases are regulated by different interleukins. In the early phase, in addiction to IL-3, there is the interaction with IL-4 and there is the involvement of other adhesion molecules such as VCAM and Stem Cell Factor (SCF). In the late phase, the most important cytokine is IL-5. This step is fundamental because the rearrangement of the heavy chain occurs. During the Pro-B stages of antigen independent maturation of B cells because the cell never encountered the antigen, bone marrow stromal signals trigger BCR gene recombination and in particular the rearrangement of the Igµ (heavy chain) genes. For this reason, the pro-B phase is crucial, and a successful rearrangement will lead to a huge proliferation. 6 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 After receiving the signal from the bone marrow, the cells can start opening their chromatin. The signal received will activate the transcription factors Pax5 and E2A and the heavy chain’s gene rearrangement will begin. The heavy chain formed must be then tested and the mechanism to do so is similar to that of the β chain of the TCR. The heavy chain is associated with an invariant surrogate light chain to test if the heavy chain is working or not. The surrogate L chain is composed of two segments: VpreB and Lambda5. 1. Thanks to the spontaneous dimerization of the pre-BCR we have the start of the signalling; 2. The dimeric BCR interacts with the ligand expressed by the bone marrow stromal cells. 3. This interaction will cause a signalling that is mediated by the Bruton thyrosine kinase (BTK); 4. Allelic exclusion and block of H chain gene rearrangement and consequently cell proliferation; The last step happens because the signal went through, and this means that the heavy chain is working correctly and thus there is no need for gene rearrangement. 7 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 If the heavy chain fails to deliver the signal, the B cell will rearrange again the residual genes. If this is not possible the B-cell will then rearrange the gene on the other chromosome. If the cells fail to deliver the signal again, it will undergo apoptosis. Children with an inherited defect of BTK have no B cell and no antibodies. (Bruton Disease) These children suffer from α-gammaglobulinemia. After the signal of the Bruton Thyrosine Kinase go through, the cell can undergo proliferation and the resulting pre-B cells can start the rearrangement of the light chain. L chain recombination is driven by IL-4 and IL-5. During this phase interaction with adhesion molecules is maintained and CD-19 is still present. The light chain formed after the gene rearrangement, will substitute the surrogate one composed of VpreB and Lambda 5. 8 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 The presence of functional chains stops the rearrangement of the other genes including the one present on the other chromosome (Allelic exclusion). These checkpoints are crucial for the formation of a functional BCR. The presence of a functional cytoplasmic µ (heavy) chain leads to: a. inhibition of other H gene rearrangement; b. allelic exclusion (transgenic mice for the µ chain do not rearrange endogen genes); c. induction of L chain rearrangement where NFkB plays a critical role; d. isotypic exclusion of L chain allele (k or λ); Heavy chain rearrangement First there is the rearrangement of the μ (heavy) chain. If it’s functional we will have the synthesis of the Igμ chain and cell can start the rearrangement of the light chain. If the first rearrangement is not functional, the cell can try the rearrangement of the second allele of the μ chain. If also the second rearrangement is not functional, the cell undergoes apoptosis. If it is functional, the cell can proceed to the rearrangement of the light chain. 9 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 Light chain rearrangement In human we also have the possibility to rearrange k and λ chains (60:40 ratio) for the light chain. 1. First the B cells try to rearrange the k chain 2. if it fails to give rise to a functional chain, there is the rearrangement of the other k allele 3. If it doesn’t work, there will be the rearrangement of the first allele of the λ chain. 4. If this also doesn’t work, there will be the rearrangement of the second allele of the λ chain. If any of this step succeed, there will be a functional BCR on the cell surface and the inhibition of other gene rearrangement. Otherwise, the cell will undergo apoptosis. apoptosis Heavy and light chain rearrangement 10 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 After a successful pre-B phase, the resulting cell is an immature B cell possessing functional BCRs made of IgM and the CD-19. A mature virgin B- cell, however express BCR made by IgM but also by IgD, all with the same specificity. CO-EXPRESSION OF IgM and IgD Mature follicular B cells (FoB2) display IgM and IgD co-expression due to a differential polyadenylation and splicing of their primary mRNA rather than DNA rearrangement. The segment VDJ can bind a constant μ region and have an IgM. The VDJ segment, however, can bind the constant δ region and have instead an IgD. Thanks to this phenomenon we have the possibility to have on the surface both IgM and IgD. The in vivo role of IgD Mature B cells display IgM and IgD but when transformed in plasma cell they never secrete IgD. Which is the in vivo function of IgD? It was observed that IgD on self-reactive B cells capture endogenous antigen but fail to transduce signals and to become antibody secreting cells. The function of IgD is to capture endogenous antigens. Negative selection of the BCR Immature B-cell have a lower expression of adhesion molecules and can thus migrate toward the periphery. However, these immature cells undergo to a mechanism of negative selection of the BCR. This happens because B cells that have finally acquired their definitive individual BCR are apoptosis prone cells. Their survival will depend on the strength of the signal delivered by the interaction between their BCR and bone marrow cells. Cells possessing a non-functional or self-reacting BCR cause cell apoptosis. Receptor editing B cells that leave the bone marrow must not react against self-antigen but must work correctly. Sometime a not desired BCR is assembled on a B cell (autoreactive). Therefore, Ig gene rearrangement continues, especially that of the V regions in the L chains, to obtain a functional and not autoreactive receptor. This is the last possibility for the cell to have a functional BCR and this phenomenon is called receptor editing. 11 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 B CELL DIFFERENTIATION After the maturation of cell, we will have different type of B-cells: B1 cells that possess the CD5 on the membrane and a BCR made of IgM; Transitional B cells that have an IgM BCR and have a receptor for BAFF that is very important for its survival; B2 cells (Derived from transitional cells) that possess both IgM and IgD BCR; Marginal Zone B cells, that possess just IgM; Follicular B2 cells that possess BCR made of both IgM and IGM; In the periphery we will have: B1, Marginal Zone, and Follicular B2; B1 Transitional B2 Marginal Follicular Zone B2 BCR IgM IgM IgM IgM IgM TYPE IgD IgD B1 cells; CD5 B cells (IgMHigh IgDNull CD5+) B1 cells arise early in embryonic development maturing both from liver and bone marrow; B1 cells are 5% of all B cells; B1 cells home predominantly in peritoneal and pleural cavities where they are self-renewing; Their BCR has a limited diversity as lack TdT; TdT is a very important enzyme that create BCR variability CD5 is a marker of this B cell population; CD5 is a scavenger transmembrane receptor that: - Enhances IL1 signals; - Promotes B cell survival; - Enhances the transduction of activation signals; 12 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 B1 cells mainly produce IgM directed to polysaccharides, microbial lipids or oxidated lipids and to red cell antigen (AB0). They produce natural antibodies; In the lamina propria half of the plasma cells derived from B1 cells and mainly produce IgA; B1 cells have been also described in humans but surface markers are no specific. All the information we have derive from mouse models. Marginal Zone; (MZ) B CELLS (IgMHigh IgDNull) B2 MZ B cells are a minor population that derives from B2 cells. They localize in the outskirts of follicles, mainly in the spleen. The interaction of Notch receptors on their membrane with the Delta ligand expressed by endothelial cells and cells of the red pulp of the spleen, induces their commitment toward B2 MZ cells. Their homing in the marginal zone of the follicle is guided by chemokines and integrins of stromal cells. Their survival and activation rest on BAFF, a TNF-family cytokine produced by Dendritic cells, follicular Dendritic cells, T cells and Macrophages. MZ B cells express CD1d antigen, which is particularly important in the infection of mycobacteria. CD1 binds glycolipid antigens and presents them to invariant NKT cells. MZ B cells express the complement receptor CR2(CD21) that is able to amplify their activation. This underly the cooperation between the innate immune response (complement cascade) and the adaptive immune response (Marginal zone B cell). Immunohistochemical staining of the follicle in which the brown cells are the CD20 positive. CD20 is a marker used to analyse B cells. At the margin of the follicle (outer circle) Marginal Zone B cells are visible Follicular B2 cells; FoB2 B cells (IgMhigh IgDhigh) Are the majority of B cells in adults; Develop in the bone marrow; The absence of Notch 2 signalling favours the commitment of transitional B cells towards FoB2 B cells; 13 Marco Sanfilippo/Eleonora D’amore – Lesson 10 – Immunology (Prof. Claudia Curcio) – 4/11/2021 The production begins just before birth and reaches significant levels only after the first year; Circulate in the bloodstream and lymph and localize in the follicles of the spleen, tonsils and lymph nodes; Compete to enter the lymphoid follicle where they receive survival signal through BAFF>BAFF receptor interaction; FoB2 B cells display a very large (> 1011) repertoire of different BCR binding sites; A virgin mature FoB2 B cell requires T cell collaboration to be fully activated, proliferate and produce antibodies; o As seen in severe combined immunodeficiency, absence of mature T cells affect also maturation of B cell, in particular FoB2; Once activated FoB2 B cell produce IgM first, and then display the isotype switch driven by TFH cells; Undergo somatic hypermutation and clonal competition resulting in a high affinity antibody production. 14 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 T CELL ACTIVATION Summary of the previous lessons T cells maturate in the thymus; B cells maturate in the bone marrow. The negative selection of T cells consists in the lack of a TCR function (so maybe the beta chain is not correct and so on), so they will die. Instead, the positive selection is due to the MHC. So, a big difference between T and B cells is that in the positive selection of the T cells, the APCs (that can be epithelial, medullary or cortical thymic cells, or even dendritic cells and macrophages in medulla) present cell peptides in the pocket of MHC class I and II, whereas B cells recognize the antigen directly. So, they can recognize some self-antigens when present in the bone marrow, and if the BCRs are binding with strong affinity self-antigens they will die, as well as with very low affinity because they do not receive enough survival signal to exit from the bone marrow. Instead, steps in common between T and B cells are: 1. Proliferation: cells undergo proliferation, in a very small way in the beginning, due to cytokines in the thymus or in the bone marrow; 2. Rearrangement (somatic recombination of the receptor) of the beta chain for T cells or the heavy chain for B cells; 3. Check if the chain is working: both can be bound by an invariant chain (alpha for TCR, light chain for BCR). This is important because it gives a strong signal for the second wave of proliferation. In this signal, especially for the B cells, it should be mentioned an important kinase which mediates proliferation, allele exclusion, and rearrangement of the light chain: the BTK. If absent, people are immunodeficient, unable to perform an efficient maturation of B cells. This condition leads to Agammaglobulinemia because they are not able to produce antibodies. B and T cells mechanism of maturation is a bit different, because T cells need to be selected positively to bind the HLA in order to recognize it, but they should bind with low affinity what is inside the pocket of HLA, otherwise it would be autoreactive, provoking big danger in periphery. On the other hand the B cell, with the rearrangement of the light chain has a BCR that can recognize the native antigen present in the bone marrow. Another big difference is that a T cell exiting from the bone marrow is mature: nothing changes on the TCR, composition and so on. Instead, a B cell that exits the bone marrow is immature because it can still change and acquire some typical features of the three subpopulations (B1, B2). In the B2, depending on the presence or not of Notch signal, we have mantellar (or marginal) zone B cells, or the follicular ones. In this lesson it will be discussed what happens in the periphery with T cells. There are a lot of T cells exiting from the thymus because there are two mechanisms impacting on the difference of the TCR: 1. Rearrangement: it provides a repertoire of millions; 2. Junctional diversity (during the recombination): it generates a hypervariable region that will bind the antigen. So, there is a huge number of T cells in periphery. We suppose that a T cell in a lymphoid organ (for example a lymph node) is recognizing an antigen. The first step after the antigen recognition is the activation of proliferation, so the T cell gives a clone, an effector T cell. 1 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 An effector T cell: Increases the number of effector cells; Kills infected, damaged or transformed cells; Induces the production of antibodies; Drives a more efficient inflammatory response, involving both innate and adaptive cells. Two different types of T cell activation are present: 1. ACTIVATION OF NAIVE (OR VIRGIN) T CELLS 2. ACTIVATION OF THE EFFECTOR CELLS ACTIVATION OF VIRGIN T CELLS Virgin or naïve T cells exit from thymus, go to the periphery and encounter for the first time the antigen. Their activation is a complex process and can be also dangerous. In fact, the presence of too much antigen can make T cells exhausted or even kill them, and so they can actually die during activation because too strong. Or we can have the opposite effect: T cells cannot respond to the antigen, and so they become anergic. So, the activation process needs to follow specific steps, and requires specific features in a way that, without one of them the activation cannot be efficient, resulting in a dead or anergic T cell that can’t become an effector. So, for this process, specialised cells for the antigen presentation are required. These are dendritic cells because, besides presenting the antigen and the HLA class I or II, they are the only ones which can perform cross-presentation: an exogenous antigen is presented with class I. So, they express the costimulatory signals which are mandatory for the full activation of virgin T cells. Why is it so important the presence of so many steps? For example, there are so many T cells in lymph nodes, very close to each other: it means that a dendritic cell with its long dendrites can connect and activate many T cells simultaneously. Thus, it is necessary that only very specific T cells for the antigen are activated, otherwise it would result in aspecific activation that can be dangerous. 2 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 The previous image shows the frequent route of entry of pathogens (viruses, bacteria, parasites etc) through skin, mucosa of the gastrointestinal tract, and the respiratory tract. There are a lot of phagocytes and dendritic cells which, after the phagocytosis of the antigen, can migrate into the lymphoid organ (lymph node, or spleen or lymphoid organ associated to the mucosa) where they present the antigen to virgin T cells concentrated in the T area. The antigen can also enter the blood by itself and in this case, it preferentially goes to the spleen thanks to a lot of afferent vessels and important arteries. Here, a section of a lymphoid organ is represented: through high vessels, dendritic cells present the antigen (in pink) to T cells (in brown) in the T area, while B cells are activated by the antigen that is coming from the lymph or from the blood. Dendritic cells arriving in the lymphoid organ can present the peptide to class II or even class I, thanks to cross presentation. Each of the T cells present in the lymphoid organ try to scan the surface of dendritic cells, looking for peptide-HLA complex that is specific for its TCR. Remember that all T and B cells in the periphery are usually prone to apoptosis, so they are really looking for activation and so, for the right complex. When they find it, they start to become more attached to the dendritic cell. In order to do this, both cells start to express more integrins, adhesion molecules and their ligands, building an immune synapsis, a small area of contact between the two cells in which there is an exchange of signals from both of them. The synapsis is called SMAC (Supramolecular Activation Cluster: supramolecular because a lot of molecules are involved, activation because it only occurs if the T cell has specific receptor for the complex, and cluster because there is a sort of clusterization of many molecules in a specific area of both T cells and APCs). The dendritic cell is bigger than T cell, so it can do SMAC with more than one T cell simultaneously, whereas a T cell can be engaged only with one dendritic cell. 3 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 Both T and dendritic cells express molecules such as ligands, integrins, or adhesion molecules like ICAM. When TCRs recognize a more specific complex, they change the conformation of ICAM (for example) from low to high affinity, and more molecules are recruited in this area, which is important for forming stronger bonds between T cells and the APCs. So, many molecules cluster the area of the SMAC, instead of being randomly present on the surface of the cell. The TCR needs 36 hours to signal inside the T cell, so it’s not so fast the activation. Thanks to the conformational changes of ICAMs or adhesion molecules, the SMAC can maintain the contact between the two cells, so there is the time to perform all that is needed for full activation. In the TCR, the beta-alpha chain contacts part of the HLA and part the peptide. The strong complementarity of the binding is due to the CDR, the complementary determining region of the TCR, in particular, the hypervariable region contacting the antigen, whereas the other CDRs contact the HLA molecule. Moreover, alpha and beta are not able to signal inside, so they are connected to CD3, a supramolecular co-receptor made by different chains (ε, δ, ζ) that are connected to the TCR and possess a cytoplasmic tail with a lot of ITAM motifs (the sequence in which tyrosine can be phosphorylated and acts as a dock for other kinases or molecules). When the TCR is recognizing the complex, it’s changing the conformation, and this change is translated to the CD3, which can be phosphorylated in the ITAMs and trans-phosphorylate the ITAM motifs in its chains. However, TCR needs to detach and attach from and to the complex a lot of times to transfer this information to the CD3: it is called piston movement. The phosphorylation of ITAMs is possible because during the piston movement there is the CD4 or CD8 co-receptor binding the HLA molecule, acting as a rope which maintains the TCR close to the complex, then the TCR can move. CD45 is another co-receptor involved: it is a phosphatase and is expressed on all leukocyte cells. In the case of T cells, it removes the phosphate group from the Src kinases, especially FYN, LCK, LYN, that in steady state are usually phosphorylated. In this way, after the piston movement, the CD45 removes the phosphate from these kinases, activating them. Then, they can phosphorylate the ITAMs in the CD3 chain (especially the zed chain, which is the one with more ITAM motifs ➔ at least 4/6, whereas the other only 1/2). 4 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 Then, the phosphorylated tyrosine becomes the dock for other important molecules involved in the activation: ZAP-70 which phosphorylates other mediators of the activation and signalling, called LAT and SLP-76. In the image: the lipid raft, an area in the plasma membrane, changes its composition in phospholipids and lipid; when the TCR is changing conformation, the CD45 recruited in the SMAC area removes the phosphate from the Src kinases ➔ from inactive (black) they become active (orange) and can phosphorylate ITAMs on CD3.Then, phosphorylated ITAMs bind ZAP-70, another kinase that phosphorylates SLP-76 and LAT. So, these are all mediators that start the pathway signalling for the activation of T cells. There are three different pathway scenarios: 1. Ca pathway: The activation of LAT and SLP-76 can activate the Phospholipase C Gamma and this, in turn, activates the phosphorylation of PIP2 in PIP3 and the separation of DAG. The PIP3 is important for the opening of Ca2+ channels on the cell surface: a lot of Ca2+ enters the T cells where it can modulate the cytoskeleton, very important for the next step, the division of the two daughter cells. The increase of Ca2+ brings to its binding with calmodulin, which activates calcineurin, that is responsible for the de-phosphorylation of NFAT-C. This is a transcriptional factor that in this way can move into the nucleus where it mediates and activates the transcription of specific clusters of genes, such as cyclin genes, kinase cyclin dependent genes ➔ genes necessary for proliferation and, subsequently, the maintenance of clonal cells. 5 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 2. PKC mediated pathway: The DAG can activate the PKC which can phosphorylate the inhibitor usually bound on NF- kB. NF-kB is one of the master regulators of inflammation, because it induces transcription of a lot of cytokines and chemokines genes, and the activation of innate cells. Usually, a steady state is maintained in the cytosol, in which NF-kB is bound to an inhibitor. When the PKC is activated, the inhibitor is phosphorylated, so it detaches from NF-kB (and then is degraded by the proteasome) that can move in the nucleus where it starts the transcription of new genes involved in mitosis or in the transcription of cytokines. 3. RAS mediated pathway: LAT is always recruiting GEF, that can activate RAC, RAS and RAF (all the RAS member proteins), which will phosphorylate cJUN and cFOS which join in AP1, the transcriptional factor that moves into the nucleus and starts the transcription of a set of genes necessary for the activation. 6 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 All these pathways can be simultaneously activated. To summarize: After the TCR recognition of the HLA-peptide complex, the CD45 activates protein kinases through their de-phosphorylation, which leads to the activation of the phospholipase-c-gamma that produces PIP3 inducing the increase in calcium, and so the activation of NFAT-C, or it produces DAG that mediates the activation of PCK and, consequently, of NF-kB. On the other hand, LAT always activates the RAS proteins and so MAPKs which phosphorylate cJUN and cFOS which join in the AP1 transcriptional factor. This is what is called the “forest of signals” which mediates the activation of T cells, but this is only a part of the signal, because these are pathways related to the TCR recognition. So, TCR signalling is not sufficient to activate a virgin T cell (this is the main difference between virgin and effector cells). ACTIVATION OF THE EFFECTOR CELLS Clonal cells produced after the activation of virgin cells differentiate in effector cells, which are not trapped in lymphoid organs (or they would not be able to work in the sites of infections like the skin, lung, intestine), but are going to the periphery, reaching the site of entry of the pathogen, where they recognize and activate the antigen again. So, like virgin T cells they are activated in the lymphoid organ, but then move to the periphery and there the only TCR signal is sufficient for their full activation. This is not true for virgin cells which also require co-stimulatory signals, so signals coming from other molecules present in the SMAC. There are two big categories of co-stimulatory molecules: 1. Ig superfamily: It includes B7 ligands (CD28 or CDLA-4) binding B7, and ICOS ligands binding ICOS receptors. Receptor and ligand are expressed, one on T cell, and the other on the APC, or viceversa. For example, B7 is expressed on the APC whereas CD28/CDLA-4 on the T cell. The same is for the ICOS. TCR, HLA, and molecules belong to Ig superfamily where the binding is mediated by the complementarity of the globular domain. 2. TNF family: It includes OX40 with OX40 ligand, 4-1BB, CD27, CD30, CD40. As for TNF receptors, these molecules need to trimerize and bind to their ligand to signal. In this image there is the CD28 on the T cell binding the B7 on the APC. B7 is also called with the new nomenclature CD80 (B7.1) or CD86 (B7.2). This is the first co-stimulatory signal important for the activation. Usually, dendritic cells express constitutively the B7 on their cell surface (though not at huge levels), so after the activation of TCR, T cells start to express the CD28, so one of the genes expressed by TCR activation is also CD28. This binding is important to induce the expression of another molecule important for T cell activation, the CD40 ligand. 7 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 Different T cell populations have a different dependence from this co-stimulatory signal (CD28-B7 binding): For naive T cell activation, it is mandatory, while for the effector cells much less, and for memory cells is not necessary at all. The reason is that the memory cells are circulating in lymph nodes, spleen but also in the periphery because they are patrolling everywhere and so, they must require less steps for full activation to have a quick activation and so, a quick efficient immune response at the second or successive encounter of the pathogen. CD4 is more dependent than CD8 on this binding. Regulatory T cells (controllers of the immune response) are dependent on this binding like the CD4. The first binding of CD28 with B7 is important to induce the expression on the T cell of the CD40 ligand, important also to mediate the full activation of B cells from T cells, and in the adaptive immune response. The CD40 ligan binds to CD40, which belongs to the TNF family (so it’s a trimer, like its ligand). It is important because the CD40 in the APC is inducing the expression of more and more CD80/86. This means that the first binding of B7 with CD28 induces the CD40 ligand to ask the APC more co-stimulatory molecules, in order to have fully activated cells and not anergic. If during activation some of these co-stimulatory molecules are missing for any reasons, (like genetic reasons in which immunodeficient people don’t have for example the CD40 ligand, B7, CD28 or CD40 is not working) T cells become anergic (even if they receive all the signals from the TCR recognizing the antigen and the complex), so they are not fully activated. The CD40 ligand is important to increase the CD80, so other co-stimulatory molecules. They are for example the OX40 and the CD40 ligand expressed on APCs that induce the expression of IL-2 through the activation of NF-kB. In general receptors belonging to TNF superfamily induce apoptosis, but not in this case, because they are binding inside TRAF (TNF receptor activation factor) and not TRADD which is the TNF receptor apoptotic domain, required for the induction of apoptosis. So, this induces the activation of NF-kB which, in turn, induces the transcription of genes in the nucleus. At this point, one gene in particular is very important and is the third necessary signal for the full activation of T cells: the IL-2. Its transcription, which starts a few hours after the contact between T cells and APCs, is important because it is supporting the proliferation (IL-2 is a sort of growth factor for the T cell). Indeed, T cells start to express with an autocrine effect the IL-2, which supports their clonal expansion. To summarize: 1. Firstly, the adhesion molecules are building the SMAC; 2. Recruitment step: changes of the plasma membrane and the formation of lipid rafts in which are recruited a lot of molecules (TCR binding and recognising the complex is activating all CD8, CD4, CD45 etc signals) 3. Especially the recruitment of CD28 on T cells and CD80 on APCs is important because it’s the first co-stimulatory signal that induces the expression of CD40 ligand in the T cell that binds to CD40, inducing the expression of other CD80 molecules from the APCs. All these events require a quite long period: to become effectors, T cells need at least a couple of days, and if the interaction is shorter, they can potentially become anergic. So, the co-stimulatory signal is important to enhance the TCR signalling, and increases the survival of T cells, allows clonal expansion and cytokines secretion (especially IL-2), and triggers the initial differentiation, because at the end of the process there will be the differentiation of CD4 in all subpopulations and of CD8 in cytotoxic. Without it, T cells become anergic or die. 8 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 In the following image a SMAC is represented: after the first contact with the APCs there is a different recruitment of adhesion molecules, TCR, co-stimulatory molecules and inside the CD3. All the scatter molecules are present around. In very few minutes a different recruitment of all these molecules occurs, to render the contact more efficient with APCs. In this picture made in the microscope the T cell is stained with CD3, which is concentrated in the synapsis. LFA-1 is instead concentrated in the APC. The green signal is made by the overlap of the two signals because they are present in the same area: this is important to render the contact efficient. The first signal is the TCR signal with all the activation signals, while the second is the co-stimulatory signal with the activation of the transcription of different genes. The co-stimulatory is inducing the transcription of IL-2 and another important molecule which is ILRα (they represent the third signal for the full activation), the receptor of IL-2 in which the alpha-chain is the one increasing its affinity for IL-2. Each T cell produces IL-2 which is secreted in an autocrine way and is in the middle of a crowdy population of T cells, so, even the closer ones can use the IL-2. Though, T cells which express the alpha-chain on their ILRα are advantaged compared to the others, so even a few molecules of IL-2 are immediately bound by the receptor, which has increased its affinity. 9 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 On the other hand, non-activated T cells need a lot of IL-2 to bound to its receptor and so, to activate the signal inside, and this represents a disadvantage. After these 3 important signals the proliferation of the cells that recognised the peptide-HLA complex occurs, along with the expansion of T cell clones which can differentiate in effectors and/or memory T cells which go to the periphery to face the infection of the pathogen. When we discussed the cytokines receptors, in particular the ones for IL-2, we also said that not all cells are dependent on IL-2 for their expansion. For example, CD4 T cells are more dependent, whereas CD8 T cells need less amount of IL-2, increasing 50.000-fold their proliferation. During a viral infection, in which CD8 are the major effectors because they can directly kill the infected cells, we can even reach up to 10% of solely CD8 clonal cells in our blood. Usually this percentage is much lower (it reaches 15% when we have a lot of CD8). The production of IL-2 can also induce the expansion of the “bystander cells”, the T cells that surround the activated cell: in this case they are not specific for the antigen, but can survive and undergo a few rounds of proliferation just using the cytokines secreted by the activated T cells. So, what we have from an infection is a great expansion of CD8 and a minor one of CD4, both recognising the antigen. Following image: the peaks show that the maximum expression is reached after 10 days, so it is important to keep this in mind when facing an infection because at the first encounter of the pathogen some time is needed for the full activation and clonal expansion of T cells. Anyway, after the clearing of the pathogen the phase of contraction occurs, because a high number of clones cannot be maintained since we don’t have such a huge space in our blood or bone marrow, or even in our organs. For example, if you have sore throat, lymph nodes are bigger, meaning that T cells are expanding clonally. If T cells don’t decrease in number, lymph nodes would become increasingly bigger after many encounters of pathogens, and this is not possible. So, during the contraction phase the number of effector T cells will decrease, but remains higher than the beginning, because some of them become memory cells. Usually, the number of memory T cells is higher than the number of the same virgin T cells, therefore, they respond faster to a subsequent encounter with the same pathogen. 10 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 So, during the expansion phase differentiation occurs: some T cells become effectors, others memory cells. This is true for both CD4 and CD8. CD8 can differentiate in cytotoxic, whereas CD4 can differentiate in many subpopulations: T helper 1, 2, 9, 17, or T regulatory cells ➔ they differ from the type of cytokine they produce which is important to modulate the response to the antigen. A specific pathogen induces a specific subpopulation because specific cytokines are necessary to induce specific antibodies, or to modulate the activation of specific innate immune cells. Scientists are still trying to elucidate if memory T cells are generated from the effector T cells, or if they are immediately differentiated from virgin T cells. 11 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 MEMORY T CELL PROPERTIES What is different in memory T cells? 1. They respond faster to the antigen; 2. They are more abundant than the naive; 3. They migrate to the tissue, where they are found when they encounter the same antigen, so they do not necessary be in the lymphoid organ to be activated (on the contrary of virgin cells); 4. They express higher levels of anti-apoptotic molecules (for example Bcl-2), because they are prone to apoptosis, but must survive. Some survival signals are coming from cytokines as for the bystander cells so, from the ones produced during the activation of other T cells. Other survival signals are coming from the crossmatch with a similar antigen. In this way, even if their TCR is bound to a complex which is not exactly the specific one, but a very similar one (which maybe changes in only 1 aminoacid because many pathogens are very similar to each other) it constantly, even if slowly, induces proliferation, maintaining a certain number of them. If we go under this threshold, even if they are memory cells, it’s like starting from the beginning, and so they need 7-10 days to perform a real response. Summarizing… The first signal for the full activation of a virgin T cell derives from: 1) The encounter of the HLA-peptide, that is driven by all the chemokines and cytokines that are driving the circulation of naive T cells in the lymphoid organs. 2) The avidity of the TCR for the complex presented by the APCs. This avidity is very high because it’s needed for the piston movement. 3) Some bounds important to increase the stability of the contact between T cells and APCs. The piston movement can increase even 100 times the response of T cells. And so, this determines the duration of the binding (long lasting is better). 4) The duration of the binding 5) The number of TCR expressed on the T cell surface. Usually, when they exit from thymus, they have a lot of TCRs, but it has been demonstrated that, for the virgin cells, they have to interact with at least thousands of HLA complexes. So, the first signal is driven by specificity. Then effector T cells need to be activated, so the full activation needs to be induced with co-stimulatory signals. Then, effectors have to decrease in number. CONTRACTION PHASE Effector cells can decrease in number thanks to different factors, in particular CTLA-4, that is the same ligand of CD28. It binds CD80, but CTLA-4 is expressed only after the activation, when the effector T cell has already cleared the pathogen. The CTLA-4 (cytotoxic T leucocyte antigen) has higher affinity for CD80 than CD28. It mediates the apoptosis of effector cells. So, the CTLA-4 switches off the activation of effector cells. The other important molecule is CD95, that is the Fas receptor: many cells, especially in periphery, can induce the Fas ligand and so, the apoptosis of effector cells. 12 Eleonora D’Amore/Beatrice Festa Bianchet – Lesson 11 Immunology (Prof. Paola Cappello) – 16/11/2021 Another important mechanism is the upregulation of regulatory T cells. T regulatory cells are induced in different conditions. They can be generated in the thymus when they have high affinity for the self-peptide, or in periphery in the presence of specific cytokines. They are important to maintain the homeostasis of the tissue, so they maintain the right number of T, B and other cells through secretion of anti-inflammatory cytokines. This is very important to switch off the inflammatory response induced by the innate, but also adaptive response. Another molecule important for the contraction phase is IDO (indoleamine dioxygenase), an enzyme that depletes the tryptophan from the microenvironment, an amino acid important for the survival of T cells and expressed by anti-inflammatory macrophages. The last factor: effector T cells start to express many anti-inflammatory cytokine receptors, so they can sense the anti-inflammatory cytokines, such as IL-10 and TGFβ, mainly produced by the regulatory T cells or the anti-inflammatory macrophages. These are all mechanisms that allow the adjustment of the activation of T cells and so, the correctly performed contraction phase. CTLA-4: why is it an inhibitor receptor? Because it has ITAMSs, so when it binds the CD80 on the APC it recruits the phosphatases instead of kinases. So, the phosphatases eliminate all the phosphates from the ITAM motifs of the CD3 co- receptor, switching off the signal coming from the TCR. In this way, it stops the expansion in lymphoid organs. (This will be discussed in the immune response in the presence of tumor) There is another ligand similar to CTLA-4 with the same function, but it acts more in periphery instead of lymphoid organs: PD-1. This ligand and all the other processes we discussed, such as differentiation of Treg, the appearance of Fas ligand and the anti- inflammatory cytokines secretion, are more important in the periphery, where they switch off and kill the effector cell. In some cases, especially in experimental models, the administration of soluble CTLA-4 has been demonstrated to completely block and inhibit the expansion of T cells against an antigen. The experiment posed the basis for the concept that we can exploit therapeutically the CTLA-4, for example in organ transplantation because in this case we need to maintain T cells suppressed, not able to react against the HLA complex. So, you administer different suppressor drugs in therapy, including the CTLA-4, but also inhibitors of the IL-2 receptor and inhibitors of other pathways activated by the TCR. 13 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Summary and continuation of the previous lesson The past time we discussed about the three important signals necessary for T cell activation, in particular for naïve T cells. The first signal, mediating the specificity, is TCR activation The second one is made by co-stimulatory molecules The third one is the IL-2 In different conditions there can be different types of activation. We discussed in detail the activation of virgin T cells and that TCR activation always occurs, even for effector and memory cells. DIFFERENT T CELL ACTIVATORS There are different situations and types of activation based on the kind of antigen: Oligoclonal activators, such as the HLA-peptide complex. In this situation there is the activation of very few T cells because they need the specific TCR for the complex. Polyclonal activators, such as mitogens which are the substances that induce mitosis and so, proliferation. For T cells these are lectins (ex: concanavalin, phytohemagglutinin) that bind TCRs not so specifically like the HLA-peptide complex but are enough to cross-link many of them (remember that a T cell needs to bind thousands of TCRs to be fully activated). Because lectins are huge molecules (they are recognised by the PRR of the innate immune cells) they can cross-link, so, bind many TCRs simultaneously and induce activation. Though, because this link is not so TCR specific, they can actually activate much more than what a single HLA-peptide complex does. So, these molecules are called polyclonal because they activate the proliferation of many T cell clones, while oligoclonal activators only a few. In lab we can also exploit two antibodies to obtain polyclonal activation (the activation of many T cells): the anti-CD3 and anti-CD28. Indeed, conformational change of TCR is transmitted to CD3 that in this way can be activated; and then you need CD28-B7 binding. So, if you exploit an antibody against CD3 this will bind all CD3 of all T cells, so many clones can be activated. Using anti-CD3 with anti-CD28 you also reach the activation of virgin T cells because you mimic the TCR signal with the anti-CD3, and the co-stimulatory signal with anti-CD28. Then, there are some chemical reagents: phorbol and ionomycin. They can be used to obtain polyclonal activation because they trigger Ca signal and kinase activation, reaching almost the three pathways activated by TCR. They are used in lab to activate T cells, because you are for example studying the immune response against an antigen, trying to figure out if they can release some cytokines, or supposing they are anergic. Because they make such a huge signal for activation, you should see the release of cytokines, independently of the specificity of the antigen. Superantigens that can activate a lot of T cells and are more similar to normal antigens because usually they come from viruses or bacteria. The number of T cells activated by the conventional antigen (HLA-peptide complex) is 1/105 -1/108, by mitogens is practically all, whilst by superantigens is usually 1/10 (5-25%). So, even if they are similar to normal antigens, they can activate more T cells than them. 1 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Superantigens are: Staphylococcal toxins Streptococcal exotoxins Culture supernatants of Mycoplasma arthritis Exotoxins of Pseudomonas aeruginosa Yersinia antigens The M protein of streptococcus Retroviruses (endogenous) So, they are products of bacteria, or released from them (like the supernatants of a Mycoplasma culture) etc. When circulating in our body, they can activate so much T cells because they are not presented by APCs in the classical way: they bind HLA class II without exposing its pocket, so they overcome the MHC restriction because they don’t need to be recognised with a specific HLA pocket by TCRs. They can activate all T lymphocytes having a particular β chain with a specific variable segment (all T cells have different variable segments of the β chain). The Vβ 3, 12, 14, 15, 20 can strongly interact with staphylococcal enterotoxin, while the Vβ 2, 4, 8 with streptococcal exotoxin. This means that many T cells can be activated by the same superantigen because there are many of them with the same variable chains. In fact, when T cells are maturating, they recombine and produce the β chain and then go to a huge wave of proliferation. Usually, for the conventional antigen, the TCR (with CDR 1,2 and 3) is recognising the HLA- peptide complex (MHC restriction). Instead, the superantigen is bound to HLA class II, but outside its pocket and so, it can be bound by only the β chain of the TCR. Thus, the superantigen activates a lot of T cells, independently of the specificity of the TCR. Therefore, when a superantigen is recognised, there is a very strong response. This is responsible, for example, for the syndrome of the toxin (toxic shock syndrome) in which there is a huge production of cytokines due to a huge activation of many T cells simultaneously. A strong response is also systemic cytotoxicity because the TNF is cytotoxic for our cells. Moreover, immune cells start to produce a lot of anti-inflammatory cytokines to balance this storm of cytokines (a frequent reaction in young females to the tampon used for menstrual cycle) because of the presence of many endotoxins or exotoxins, mimicking a culture medium for bacteria. Because the activation and the response are so strong, T cells usually die. So, sometimes we can be deprived of a huge number of T cells and, since they are not immediately replaced, we lose part of our repertoire that can be specific for other antigens. This depletion can also happen before birth if the foetus is infected by the mother who is, in turn, infected or having the toxic shock syndrome for example. 2 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 ADAPTIVE T CELL RESPONSE We saw that after an infection or a damage of one of our barriers, dendritic cells migrate to lymphoid organs where they present the antigen to naïve T cells. After T cell recognition, activation through co-stimulatory molecules, and the presence of IL-2, a single a T cell becomes a clone which, during this presentation, starts to differentiate in an effector or memory cell. When effector and memory cells go to periphery to face an infection, they are quickly activated and so, they can perform their function by releasing certain types of cytokines, on the base of being cytotoxic, T helpers etc. Their activation is so quick because it’s triggered by any kind of cell which presents the HLA-peptide complex specific for the TCR and doesn’t require co-stimulatory signals. This quick activation is important because in this way effectors can fight against the pathogen in periphery without losing too much time, since a week has already been passed for obtaining them. So, the reaction (the pathways) elicited is 100-1000 time faster and intense than the one of virgin T cells. This is true for both effector and memory cells so, they react very fast when they encounter the same antigen a second time. This tremendous memory reaction forms the basis of vaccination. DIFFERENTIATION IN EFFECTOR/MEMORY CYTOTOXIC T CELLS Cytotoxic T cells (CTL) or T killer cells (Tc) are mostly CD8, but sometimes also CD4 because they can kill not only by producing perforin and granzyme which are typical of NK cells and CD8, but also through the release of specific cytokines: lymphotoxin and the TNF. CTLs recognise foreign peptides in the groove of self HLA class I molecules (because CD8 is a co- receptor for HLA class I) or foreign HLA molecules with self-peptides. For their activation co-stimulatory signals (ex: CD80, CD40, OX40 and so on) are not so needed, instead some particular cytokines are important, especially IL-7, IL-12 and INF-γ. In periphery, where they are fully activated by APCs, they can directly kill the target (infected cells, tumour cells and transplanted organs) without the help of any other cells. In the last 10 years it has become evident that CD4 T helper cells are very important for CTL differentiation. Of course, the presentation of the antigen to class I is not made by CD4, but by APCs (dendritic cells) through cross- presentation, though, it has been demonstrated that in the absence of CD4 T helper cells a CD8 T cell never reaches full activation so, it never enriches its cytoplasm with the typical granules and become a memory cell. 3 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Thus, these cells play a key role in the activation of the adaptive response. In particular, CD4 Th cells directly release cytokines that can be used by the CD8 T cells, recognising the antigen, for their differentiation. These cytokines can also increase, together with the CD40 ligand-CD40 binding, the efficiency of APCs to present the antigen and produce cytokines, both necessary for the CTL and memory CD8 T cells differentiation. This effect made by CD4 Th cells on APCs is called licensing. The presence of specific master genes determines the differentiation of any type of cell. The CTL full differentiation involves the expression of two transcriptional factors: Tbet and Eomesodermin (Eomes). They induce the expression of genes which are specific for the functioning of CD8 killer cells that are IFN-γ, perforin and granzyme B. Specific cytokines are required for inducing the expression of these two transcriptional factors in CD8 T cells: The ones produced by Th cells, IL-2, which is important for clonal expansion (so it’s not so specific but it’s important to induce proliferation). IL-12 and IFN-I usually produced by dendritic cells. This is why it’s important that CD4 Th cells license dendritic cells, inducing them to release IL-12 and IFN-I. These are produced during a viral infection, so CD8 T cells are important to kill a viral infected cell. IL-7 which maintains the survival of the effectors, but especially of memory CD8 T cells. IL-21 which is mainly produced by T helper follicular cells trapped in lymphoid organs. It’s important like IL-7 for the survival, especially in humans, while the IL-7 in mouse. Effector cells differentiated in lymphoid organs then migrate in periphery, where they scan the surface of our cells. They have to recognise the right HLA-peptide complex with the TCR, but after this the activation is very fast so they can immediately signal inside the cell the rearrangement of the cytoskeleton (allowed by the Ca pathway) and therefore release their granules. Thus, in periphery the TCR signal alone is enough for the activation. Other receptors can also be involved in this activation, in particular, the ones expressed by the fully differentiated CD8 T killer cells which are very similar to the ones expressed by NK cells. These receptors recognise the stress ligand or the viral hemagglutinin which are components of the envelope. After degranulation they can kill target cells. Though, besides killing with the release of perforin and granzyme, they can also do it through the expression of Fas ligand which has to bind to Fas on the target cells, or through the release of a huge amount of ATP since it has receptors, so it induces damage in target cells. They can also release the TNFα which binds to the TNF receptor on the target cells inducing apoptosis. So, in a very small area of contact between CD8 killer cells and the targets there is the release of many weapons that kill and damage the targets. This area is called the “kiss of death”. 4 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Perforins Perforin is a protein which after its release forms a pore in the membrane of target cells. This is due to its similarity to C9, a terminal component of the membrane attack complex (MAC) of the complement cascade. The MAC induces the osmotic shock in target cells. The perforin is very similar: it can introduce itself in the double layer of the membrane of target cells, where it makes a pore through which the granzyme can enter and then induce the osmotic shock (a lot of Ca can also enter through it, causing damage). Granzymes Granzymes, which stands for “granule secreted enzymes”, are serine proteases that can cleave different proteins and substrates in target cells, starting from the closer cytoskeleton proteins and then the cytoplasm ones. They can also activate the caspases, thanks to the cleavage, and DNAse which can degrade DNA. How is it possible that the CTL releasing perforin and granzyme is not killing itself? Perforin and granzyme can’t distinguish target cells from CTLs so, this is possible thanks to the expression by the CTLs of cathepsin. It is a protease which covers the inside of the granule in the CTL. When the granule fuses with the plasma membrane for exocytosis, cathepsin becomes exposed on the CTL surface, therefore, all perforins and granzymes close to the CTL are degraded by cathepsin. For exocytosis the rearrangement of the cytoskeleton is important, in particular, of microtubules which allow the fusion of the granule with the plasma membrane. After the releasing, we can also see the formation of apoptotic bodies by target cells which are then phagocyted by innate cells so, by macrophages or neutrophils. On the left you can see an image of the electronic microscope of T cells which can be very small compared to target cells, the kiss of death and the entrance of perforin and granzyme. On the right the green is to enlighten Ca flux in CD8 T cells when activated and you can see the release of granules. 5 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Here you can see a CD8 T cell detached from a target cell that has been induced to apoptosis. You can also see the pore induced by the release of perforin. Many enzymes are inactive so they need the conformational change to activate, so when the granule is a vesicle and there is no change of pH the cathepsin is inactive and can’t degrade perforins and granzymes inside. Instead, when it is exposed on the cell surface, because of the change of pH and the presence of Ca in the extracellular environment, cathepsin becomes active. Cathepsin becomes also active in the HLAII processing and presentation, when the vesicle fuses with the post-Golgi and there is a change in pH which becomes lower. One CD8 T cell after killing a target cell, can detach from it and kill another one and so on. After a while, if the antigen is persistent so, chronically present as for some chronic infections, they become exhausted, therefore, they can’t respond anymore and kill other cells. They are the most important weapon against viral disease because the infected cells of viruses start to display more virus peptides instead of self-peptides on their cell surface so, they can be recognised by CD8 T cells. Many pathogens evolve different strategies to avoid recognition and thus, their killing. Some viruses, for example the herpes viruses, can hide themselves by producing proteins which decrease or impair the expression so, the presentation, of the HLA class I molecule on the infected cells. This means that these infected cells can’t be recognised by CD8 T cells, though, they can be killed by the recognition of the missing self by NK cells which sense or not the HLA through their receptors. Besides viral infected cells, CD8 T cells can also kill: Cells infected by intracellular bacteria which induce the synthesis of bacterial proteins that will be exposed on the groove of HLA and processed by proteasome. Some of them are Mycobacterium Tuberculosis and Listeria monocytogenes. Tumour cells which, thanks to mutations inside their genes, change their proteome profile. Some of these proteins can be processed and presented in the pocket of class I and thus, recognised by CD8 T cells and killed. This happens every day for cells mutated in not so mandatory oncogenes which can be recognised as transformed and eliminated. They are also responsible for: Tissue damage in the presence of some viral infections, especially in the hepatitis B or C virus. We react so strongly against viral proteins that CD8 T cells kill a lot of our hepatocytes, so they are responsible for hepatitis after the infection. Thanks to the intrinsic ability of hepatocytes, we can replace the damaged tissue. Hypersensitive reactions, especially against some metals or bacterial/viral antigens which are very similar to some proteins of ours so, they induce the activation of CD8 T cells that can recognise our cells and kill them (in psoriasis they kill keratinocytes). So, they are responsible for the signs and symptoms of the disease. 6 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 https://www.youtube.com/watch?v=WdCiaIS2LV4: When the CTL is encountering an infected cell through its receptor, the recognition of the HLA complex, since it’s an effector cell and doesn’t need the co-stimulatory molecules, is enough to induce the release of cytokines and granules which are forming the pore through which the granzyme can enter. Inside the cell granzymes induce apoptosis. https://www.youtube.com/watch?v=Jg_21iSYwBc DIFFERENTIATION IN EFFECTOR/MEMORY T HELPER CELLS CD4 T cells differentiate in Th (T helper) cells. There are different subpopulations of Th cells: Th0 (it’s the steady state, so the naïve cell that has been engaged through the TCR. It’s called Th0 because it can secrete different kind of cytokines, thus, it’s not specialised in secreting a specific one), Th1, Th2, Th17, T regulatory (Treg), T follicular helper (TFh) etc. There are many more of them (ex: Th9, Th22). They are not CD8 T cells, even if they can release cytokines. Th cells recognise the peptide present in the complex with HLA class II because the CD4 is the coreceptor for HLA class II. Usually, Th cells, after their activation (so after the antigen presentation in the lymph node) migrate to peripheral tissues, or, they can be trapped (and this happens especially for TFh cells) in the lymphoid organ where they help the full differentiation of CD8 and B cells. The Th going in periphery, so, in tissues, help the activation of the innate immune cells, but also of CD8 T cells. There, the battle against the pathogen needs to be fight. The cytokines released by Th cells are not only important for the adaptive immune response, but also to modulate the intensity and duration of the reaction of the innate cells. The subpopulations of Th cells are different because each of them releases a specific set of cytokines. Therefore, they differently regulate both the adaptive and innate immune response. Different pathogens induce different activation in APCs and so, the expression of specific factors that will differentiate CD4 T cells in the different subpopulations. Indeed, a specific set of cytokines can clear a specific pathogen. Th0 cells, which are not well specialised in their set of cytokines, are very sensitive to cytokines released by APCs. The involvement of specific transcriptional factors determines the differentiation in different subpopulations: Th1 cells express Tbet like the CTLs, a transcriptional factor which induces the secretion of IFN-γ. Indeed, they release IFN-γ after their activation. Th2 cells express GATA3 and because of this they release IL-4, IL-5, and IL-13. Th17 express RORγt and so they release IL-17 and IL-22. 7 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Lastly, Treg cells have a master regulator gene, the Foxp3 transcriptional factor which is necessary for inducing the release of TGFβ and IL-10. It has been demonstrated, especially in vitro, that the differentiation of a subset inhibits the differentiation of another one. For example, the differentiation in Th1, and so the release of IFN-γ, inhibits the expression of GATA3, thus, the differentiation in Th2. Then, IL-4, which is released after the differentiation in Th2 cells, inhibits the expression of Tbet so, the differentiation in Th1. In laboratory, you can induce polarization of naïve T cells in Th1, Th2 and even Th17 using specific factors knowing that in the presence of a subset, you can’t obtain another one. So, how can a Th0 contacting an APC differentiate in all these subsets? A crucial role is played by the APC which, based on the captured pathogen, can release different kind of cytokines, and express some ligands which are crucial in determining the differentiation of Th0 in the different subpopulations. So, everything happens during the formation of the synapsis, therefore, during activation. Th1 When facing an intracellular bacterial or viral infection, APCs can be infected by these bacteria or, if they find them in the blood, they can phagocyte them and in their presence, they usually produce IL- 12. So, when they present the antigen associated to HLA class II to CD4 T cells (= Th0), these differentiate in Th1. But, besides the production of IL-12, for the differentiation in Th1 is also important the Delta ligand which binds the Notch receptor. The Notch receptor is a “fate receptor” because, besides determining this differentiation, it’s the signal important for the lymphoid precursor to decide if remaining in the bone marrow or reaching the thymus. IL-12 induces the activation of the STAT which, in turn, activate the expression of Tbet that allows the differentiation in Th1, thus, the IFN-γ production. The IFN-γ acts on many kinds of cells and so, Th cells modulate not only the adaptive, but also the innate immune response. IFN-γ, in particular, acts on: 8 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 Macrophages making them differentiate in M1 (antiviral/antitumoral/antibacterial macrophages) which are the ones acting in phagocytosis and oxidative burst so, in the production of ROS which are important for the killing of the pathogen. Dendritic cells because it induces the expression of HLA and the basal level of co- stimulatory molecules (CD80 and CD86). B cells. The different cytokines released by different subsets of Th cells can activate the production of specific antibodies by B cells. We have different antibodies circulating: IgG, igM, igA, igA … which are important to fight a particular pathogen. The igG, which are the most abundant in our blood, are important for intracellular bacteria or viruses because they can immediately (when they are still in the blood) neutralise their entrance in our cells. For other kinds of pathogens, such as for helminths, worms and in general parasites we need to activate different responses and antibodies. For example, the igE are responsible for allergic reaction. IFN-γ induces the igG to counteract the entrance of bacteria. CTLs and NK cells because it induces the perforin and granzyme secretion. Endothelial cells increasing the expression of adhesion molecules or changing them in a more active conformation. Besides releasing IFN-γ, Th1 cells also release IL-2, needed to proliferate and survive, and TNFα. All these cytokines act on the different kinds of cells just mentioned and activate B cells to produce antibodies. IFN-γ also works in an autocrine way on the Th1 cells. In the following picture you can see a summary of all the actions of IFN-γ. In the NK cells it is inducing the release of other IFN-γ. In macrophages the burst, phagocytosis, and the expression of HLA class II molecules. In B cells the production of specific antibodies that can also activate the complement. In the CD8 the release of other IFN-γ. So, the inflammatory response induced by the release of cytokines from Th1 is important to control viral and intracellular bacterial infection. Th2 What happens in the presence of worms, parasites? 9 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 They are impossible to be phagocyted by APCs because they are too huge, though, they present on their wall/cell surface proteins, or glycosylic and glycosidic residues recognised by the PRR on phagocytes, so also on dendritic cells. Therefore, they can be differently activated (compared with the situation of intracellular bacteria) and release different kind of cytokines. In the presence of these parasites, they release IL-4: in the skin many parasites sufficiently bite the cells to induce the reaction. Indeed, in the skin there a lot of mastocytes which release al lot of IL-4 which is also released by these dendritic cells migrating in lymph nodes. Dendritic cells also express the Jagged ligand which binds the Notch receptor. So, there are only two class of ligands for Notch: Delta and Jagged. Delta is for Th1, while Jagged for Th2. The IL-4 released by APCs induces, through the activation of STAT, the expression of GATA3 which is the specific transcriptional factor for the differentiation in Th2. The Th2 cells start to produce their typical set of cytokines: IL-4, IL-5, IL-9 and IL-13. They act on different kind of cells, or, in the same ones, they induce a different activation: IL-4 and IL-13 induce the activation of macrophages in M2 which don’t phagocyte (because phagocytosis is no more necessary and so they don’t need to produce ROS to kill the pathogen inside), but release granules to eliminate these parasites or some growth factors, and even histamine that induce the muscle or endothelial reaction to help in their elimination. IL-4 and IL-5 act on B cells to induce respectively the release of igE (also induced by IL-13) and igA which can opsonize helminths, binding to their wall, and induce the activation of eosinophiles that are the only cells able to kill them because only the basic major protein can destroy their cell wall. IL-5 also induces the proliferation of these eosinophiles because they are a small population in the blood. So, in this kind of infection, haematopoiesis needs to be induced for releasing eosinophiles which, even in the tissue, need to survive a little bit longer that usual. IL-4 and IL-13 also act on smooth muscle cells and endothelial cells because you have to activate the peristalsis to eliminate these worms from intestine, or you do it with vomit, depending on where they are. 10 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 So, Th2 subset is mainly important for the fight and clearance of this kind of pathogens: parasites and, eventually, extracellular bacteria. Here there is a typical mucosa, like the intestinal mucosa, in which you can have the entrance of worms through food or contaminated water. When this happens, ? cells produce a lot of mucus because they can be trapped in it, thus, we can activate adaptive and innate immune response which can eliminate these worms. This is particularly true thanks to mast cells realising IL-4, macrophages, and dendritic cells (which activate Th2 in the lymphoid organ) that, together with the release of cytokines, allow the activation of M2, eosinophils, endothelial and master cells. This image instead shows the different Th subsets which are all characterised by a specific transcriptional factor and release of cytokines. Th17 11 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 These specifically differentiate in the presence of extracellular bacteria or fungi (ex: Candida, Aspergillus) which activate APCs to secrete a different set of cytokines. In particular, these are IL-1, IL-6 which are two important early inflammatory cytokines and IL-23, plus TGFβ. They induce, through the activation of a particular STAT protein, the expression of RORγT in the Th0 cell so, its differentiation in a T17 cell. RORγT is the master regulator of Th17 which releases mainly IL-17, but also IL-21, IL-22 and TGFβ. They, in particular IL-17 and TGFβ, induce the release of G-CSF from epithelial cells which stands for “granulocyte colony stimulating factor”. This means that in the presence of these cytokines, epithelial cells will induce the haematopoiesis to release more granulocytes from the bone marrow to the blood, especially neutrophils, but also basophils and eosinophils. Indeed, neutrophils have the receptor for IL-17 but also keratinocytes because many fungal infections are taken through skin (ex: when you are toughing a contaminated surface or going in the swimming pool). All these cytokines act on: B cells inducing the production of igA, important to activate innate cells to kill bacteria/fungi or the complement cascade since, besides fungi, also extracellular bacteria (which being in the extracellular environment are reachable by fluids, so even by the complement) need to be killed. It has been demonstrated that Th17 cells are responsible for the response to fungal infection because people defective in the differentiation in Th17 or, better, in the response to IL-6 and TGFβ, are more susceptible to fungal infection. When not properly activated, Th17 are also responsible for many autoimmune diseases because the IL-17 acts not only on macrophages and, eventually, fibroblasts which are involved in the innate response by producing cytokines or activating the inflammation, but also on endothelial cells, osteoblasts, and chondrocytes. When acting on macrophages, it induces the release of a lot of metalloproteases which cause damage in the extracellular matrix/basal membrane/interstitial tissues. So, they induce bone erosion, cartilage damage, matrix destruction and pathological mechanisms which are at the base of many autoimmune diseases, such as periodontal disease, rheumatoid arthritis, multiple sclerosis, Crohn’s disease and so on. 12 Beatrice Festa Bianchet / Francesca Fresia – Lesson 12 – Immunology (Prof.ssa Paola Cappello) – 18/11/2021 13

Immunology Lesson 10: T and B Cell Maturation (PDF)

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue