Overview of CD4+ T cell differentiation.
- 1. Lecture 3 An overview of CD4+ T cells development, differentiation , and function Michel Edwar Mickael IGHZ
- 2. Contents • CD4+ T cells branch of adaptive immune cells • CD4+ T cells activation (TCR-MHCII interactions) • CD4+ T cells Thymus selection (B selection, Effector cells, Maturation, Positive selection and negative selection) • CD4+ T cells differentiation • CD4+ T cell function
- 3. How do CD4+ T cells uniquely recognize every pathogen or foreign body entering our body ?
- 4. Father of cell mediated immunology • he discovered phagocytosis after experimenting on the larvae of starfish. • In 1882 he first demonstrated the process when he inserted small citrus thorns into starfish larvae, then found unusual cells surrounding the thorns. • He realized that in animals which have blood, the white blood cells gather at the site of inflammation, and he hypothesised that this could be the process by which bacteria were attacked and killed by the white blood cells
- 5. The adaptive responses are divided into humoral vs. cell-mediated immunity Types of adaptive immunity. In humoral immunity, B lymphocytes secrete antibodies that prevent infections and eliminate extracellular microbes. In cell-mediated immunity, helper T lymphocytes activate other cells to kill phagocytosed microbes, or cytotoxic T lymphocytes directly destroy infected cells. CD4 CD8 (Th cell)
- 6. • When professional antigen presenting cells encounter perceived pathogens, they engulf them and subject them to phagocytosis. • Then APC present these processed peptides “antigens” to CD4+ T cells through attaching them to their MHCII • CD4+ T cells use their TCRs to uniquely identify and react to presented antigens. • The area between compromising the MHC2II and the TCR is called the immuno-synapse The interaction between TCR cells and the MHCII is the hallmark of CD4+ T cells existence
- 7. Difference between antigens and Super-antigens ? Carbohydrates, nucleic acids and lipids are also potential antigens / immunogens
- 8. What are Antigen presenting cells (APCs) ? APCs present antigen to CD4+ T cells on MHC class II molecules
- 9. • Each T cell bears a TCR of one specific antigen (allelic exclusion). • There are two types of TCR receptor • The first is called αβ CD4+ T cells • It is a heterodimer consisting of α and β chains that are linked together by a disulfide bridge • Both alpha and beta two chains have a constant regions which anchors the receptor to the cell membrane • Both alpha and beta chains have a variable regions which comes in contact with the antigen and its is the regions responsible for the highly specificity of the TCR. • The majority of cells in the thymus give rise to αβ T cells, however approximately 5% bear the γδ T cell receptor (TCR). • Their repertoire is limited in comparison with alpha/beta T cells are defined by the existence of unique TCR on their surface.
- 10. The TCR exists in a bigger complex called TCR-CD3 complex • The reason behind that is the intracytoplasmic region of the TCR chain is too short on its own to transduce a signal. • The CD3 complex consists of 6 chains namely ε(2 chains) δ,γ, ζ (2chains) • CD3 complex play the major role in activating the T cell.
- 11. For complete activation to take place, two signals has to take place 1. Engagement of the TCR with the MHC2 II 2. Costimulatory molecules interaction represent the second signal CD28 on T cells interact with CD80 on APC CD40L on T cells interact with CD40 on the APC. Also other Costimulatory molecules exist such as ICOS, OX40 and 4-1BB and CD45 T cell activation pathway
- 12. Co-stimulation is necessary for T cell activation
- 13. Accessory molecules stabilizes the TCR-MHCII interaction
- 14. TCR mediated signaling 1. TCR engagement with the peptide MHCII complex initiaties the assembly of the signaling complex. 2. A protein called LCK which is associated with CD4 phosphorylates the ITAMs on the zeta chains. This cr4eates a docking site for a protein called ZAP 70. 3. When ZAP70 is activated this leads to activation of three downstream pathways that eventually leads to activation of NFAT , NFKb and AP1 and the production of IL2.
- 16. Summary Key events of T cell activation process • APC process and present a unique peptide through its MHCIII • T cell receive costimulatory signals from CD28/CD80 pathway and CD40/CD40L pathway. • Accessory adhesion molecules help to stabilize binding of T cells and APC, namely (LFA1 and ICAM1 and CD2/LFA3 pathway. • Lck which is associated with CD4 activities ZAP70 pathway. • This activates three TF pathways namely NFKB, NFAT and AP1. • These transcription factors collectively activities IL2 cytokine production • IL2 production activities differentiation and based on the eexistence of other cytokines of in its microenvironment CD4+ T cells will be pushed into a subtype.
- 17. Major Histocompatibility Complex (MHC) Molecules • T cells do not interact directly with intact antigen • T cells recognize fragments of antigen carried to the cell surface by MHC molecule • Three classes of MHC • Class I MHC are found on almost all nucleated cells of the body. • MHCI cells present peptide antigens to cytotoxic CD8+ T cells • Class II MHC are located on dendritic cells and macrophages. Their main function is to display antigens to helper T cells. • The function of MHCIII seems not to be involved with antigen presentation. • MHCIII function remains unknown.
- 18. MHCII structure • MHC class II molecules are heterodimers consisting of an α chain and a β chain. • MHCII consists of four regions • A) cytoplasmic regions which contain the sites for phosphorylation and binding to cytoskeletal elements • B) transmembrane regions containing hydrophobic amino acids • C) highly conserved B2 and a2 domains to which CD4 binds • D) highly polymorphic peptide binding region is formed of a1 and b1.
- 19. Questions for the next section How do T cells generate so much variation in the first place ? How to choose the right T cell , so the body does not attack itself? • During thymocyte maturation, 98% of T cells are discarded by selection, this is a mechanism designed to ensure that T cells function without major problems. • Variation in the TCRab gene arrangements
- 20. •Answer from the thymus •The site of T cell maturation •Thymocyte: lymphocytes in the thymus at various stages of maturation •Immature T cell lineage cells enter the cortex through the blood vessels •As thymocytes mature, they migrate toward the medulla and exit the thymus and enter the blood Where do T cells come from?
- 21. How to choose the right T cell , so the body does not attack itself?
- 22. • The earliest developing thymocytes lack the expression of the co- receptors CD4 and CD8 and are termed double negative (DN) cells. • The DN population can be further sub-divided by the expression of CD44 (an adhesion molecule) and CD25 (Interleukin-2 receptor α chain), Double negative cells
- 23. DN3 and the beta selection • DN3 are Double negative cells • No expression of CD4 or CD8 • Cells that lack expression of CD44, but express CD25 (DN3) undergo • Rearrangement of TCR beta genes (V,D,J) • beta-selection : cells that have successfully rearranged their TCR-β chain locus are selected • Based on • Loss of stem cell markers (c-Kit, CD 44) • Expression of Pre TCR (Beta chain plus pre alpha chain) • Suppression of further beta chain changes • Signal to initiate alpha chain
- 24. bTCR gene rearrangement in the thymus Db/Jb rearrangement Vb/DJb rearrangement DN
- 25. • The β chain then pairs with the surrogate chain, pre-Tα, and produces a pre-TCR, which forms a complex with CD3 molecules • This complex leads to the survival, proliferation, arrest in further β chain loci rearrangement, and further differentiation by up-regulation and expression of CD4 and CD8, these cells are termed double positive (DP) cells. B selection results
- 26. αTCR gene rearrangement in the thymus b/pTaCD3 complex: - Triggers pho-and degradation of RAG- 2 - Halting b-chain gene rearrangement (allelic exclusion) TCR b/aCD3 complex: DP
- 27. Positive Selection of T Cells (MHC restriction) • After completion of TCRα rearrangements, αβ T cells die unless they are rescued by a low-affinity interaction of the TCRαβ heterodimer with self- peptides complexed with MHC antigens that are expressed on thymic epithelial cells. • Positive selection designates T cells capable of interacting with MHC. Double-positive thymocytes (CD4+/CD8+) move deep into the thymic cortex tissue where they are presented with self-antigens. These are expressed by thymic cortical epithelial cells that express both MHC I and MHC II molecules on the surface of cortical cells. • Only those thymocytes that interact with MHC I or MHC II will receive a vital “survival signal.” Those that can’t interact will undergo apoptosis (cell death). • The vast majority of thymocytes die during this process. • A thymocyte’s differentiation into either a helper or cytotoxic version is also determined during positive selection. T cell precursor rearrangements, αβ T Immature Thymocytes Positive selection for cells that bind MHC Death by apoptosis for cells that does not bind CD3+ CD4+ CD8+
- 28. Double to Single Positive • Double-positive cells (CD4+/CD8+) that are positively selected on MHC class II molecules will eventually become CD4+ helper T cells, while cells positively selected on MHC class I molecules mature into CD8+ cytotoxic T cells. • A T cell is then signaled by the thymus to become a CD4+ cell by reducing expression of its CD8 cell surface receptors. If the cell does not lose its signal, it will continue reducing CD8 and become a CD4+, single positive cell. • But if there is a signal interruption, it will instead reduce CD4 molecules, eventually becoming a CD8+, single positive cell. • There are two theories that try to explain how Double positive become single positive cells.
- 29. Instructive model Binding precedes down regulation of non dominant marker
- 30. Stochastic model • Random down regulation occurs before binding • Nonbinders die via apoptosis
- 31. Negative Selection of T Cells • Thymocytes that survive positive selection migrate towards the boundary of the thymic cortex and thymic medulla (the part of the thymus where T cells enter circulation). While in the medulla, they are again presented with self-antigen in complex with MHC molecules on thymic epithelial cells. Thymocytes that interact too strongly with the antigen receive an apoptotic signal that leads to cell death. • Thymocytes that express high-affinity receptors for self-peptide–MHC expressed on thymic DCs are deleted in a process that is known as negative selection • Elimination of thymocytes that have TCR’s that have high affinity self MHC / bind self- MHC + self peptide ensures Self tolerance • Genes that control these processes are AIER1 and FEZF2. Self-react Non-Self-react Survival
- 34. • Some selected become T-reg cells, which retain their ability to bind to self-antigens in order to suppress overactive immune responses. • These cells may be protective against autoimmunity. • The remaining cells exit the thymus as mature naive T cells. • This process is an important component of central tolerance, a process that prevents the formation of self-reactive T cells that are capable of inducing autoimmune diseases in the host. • Autoimmune diseases reflect a loss of central tolerance in which the body’s own B and T cells become sensitized towards self-antigens. Many autoimmune disorders are primarily antibody-mediated, but some are T cell mediated. • One example of the latter is Crohn’s disease, in which T cells attack the colon. • These autoimmune disorders may be caused by problems in negative selection and tend to have genetic components. CD4+ Tregs emergence from Thymus
- 35. Where do thymocytes undergo negative selection? This has been controversial Open questions What about CD8+ Tregs? How and when they are generated? Why only two genes (FEZF2 and AIER1) control the expression of most of the proteins used for negative selection Why not every single protein is presented to inhibit the chance of auto immunity ?
- 36. Ok, now we have selected a CD4+ T cells, are there any other stages of differentiation ?
- 37. • Occurs in secondary lymphoid tissue • IL-2 levels are increased 100 times • Binds to IL-2 receptor on producing cell • Takes several days to occur T cell differentiation
- 38. Differentiation results • Functions of effectors • B cell helper • Cytokine secretion Characteristics of memory cells • Last months to years vs. effector cells that last days to weeks • Memory cells more easily activated by all APCs then naïve T cells
- 39. Memory T cells • Both CD4+ and CD8+ memory T cells can be subdivided into subsets based on their homing properties and functions. • Central memory T cells express the chemokine receptor CCR7 and L-selectin and home mainly to lymph nodes. when they encounter antigen they undergo brisk proliferative responses and generate many effector cells on antigen challenge. • Effector memory T cells, on the other hand, do not express CCR7 or L-selectin and home to peripheral sites, especially mucosal tissues. On antigenic stimulation, effector memory T cells rapidly become cytotoxic, but they do not proliferate much.
- 40. – Cytokine profile is influenced by several factors: • Nature and dose of antigen • Route of infection • Initial cytokine environment • Type of antigen presenting cell/ costimulation • Genetic background – The cytokine profile determines the effector function of the helper cell What are the aspects that control the microenvironment of CD4+ T cells differentiation ?
- 41. Cytokine effect in priming TH1 TH2 or TH17 • The differentiation of naive CD4 T cells into different subclasses of armed effector T cells is influenced by cytokines elicited by the pathogen. • Many pathogens, especially intracellular bacteria and viruses, activate dendritic cells and NK cells to produce IL-12 and IFN-g, which cause proliferating CD4 T cells to differentiate into TH 1 cells. • IL-4 can inhibit these responses. IL-4, produced by DC cell in response to parasitic worms or allergens, acts on proliferating CD4 T cells to cause them to become TH 2 cells. • They may act either when the CD4 T cell is first activated by an antigen-presenting cell or during the subsequent proliferative phase • In TH17 the antigen is extracellular fungi or bacteriaI, DC secret L-6, IL-1, and IL-23,
- 42. • Each subset of differentiated effector cells produces cytokines that promote its own development and may suppress the development of the other subsets • IFN-γ secreted by TH1 cells promotes further TH1 differentiation and inhibits the generation of TH2 and TH17 cells. • Similarly, IL-4 produced by TH2 cells promotes TH2 differentiation and inhibit TH1, • and IL-21 produced by TH17 cells enhances TH17 differentiation. Respective cytokines inhibit other phenotype Th1 Th2 IFN-γ L-4
- 43. Cytokines can inhibit respective Th cells activators • TH2 cells make IL10, which acts on macrophages to inhibit TH1 activation, perhaps by blocking macrophage IL-12 synthesis.
- 44. Antigen effect in priming TH1 or TH17and TH2 • The nature and amount of ligand presented to a CD4 T cell during primary stimulation can determine its functional phenotype. • CD4 T cells presented with low levels of a ligand that binds the T-cell receptor poorly differentiate preferentially into TH2 cells making IL-4 and IL-5. • Such T cells are most active in stimulating naive B cells to differentiate into plasma cells and make antibody. the antigen is extracellular helminth or allergen • T cells presented with a high density of a ligand that binds the T- cell receptor strongly differentiate into TH1 cells that secrete IL-2, TNF-beta, and IFN-gamma, and are most effective in activating macrophages. • In TH17 the antigen is extracellular fungi or bacteria, DC secret L-6, IL-1, and IL-23.
- 45. Antigen effect on priming TH1 or TH17and TH2
- 47. Naïve lymphocytes re-circulate until they encounter their specific antigen Lymphocytes develop from bone marrow stem cells, mature in the generative lymphoid organs (bone marrow and thymus for B and T cells, respectively), and then circulate through the blood to secondary lymphoid organs (lymph nodes, spleen, regional lymphoid tissues such as mucosa-associated lymphoid tissues). Fully mature T cells leave the thymus, but immature B cells leave the bone marrow and complete their maturation in secondary lymphoid organs. Naive lymphocytes may respond to foreign antigens in these secondary lymphoid tissues or return by lymphatic drainage to the blood and recirculate through other secondary lymphoid organs.
- 48. CD4+ T cells function
- 49. Th1 function • TH1 function • Activate CD8, macrophages and NK to do direct killing of infected cell (by secreting IFN gamma and IL-2) • Neutrophil activation • Activate B cell to secret opsonizing antibodies that increase phagocytosis
- 50. Th2 function • Bind B cell and secret IL-4 that lead to B cell activation and antibody secretion • Secret IL-5 to Activate eosinophils to react against worms • Secret IL-10 that suppress macrophages
- 51. Th2 activates B cells
- 52. • TH 1 cells activate macrophages, enabling them to destroy intracellular microorganisms more efficiently; they can also activate B cells to produce strongly opsonizing antibodies belonging to certain IgG subclasses (IgG1 and IgG3 in humans, Both TH1 and TH17 cells contribute to cell-mediated immunity, each subset serving different roles in the phagocyte-mediated eradication of infections. • TH 2 cells, on the other hand, drive B cells to differentiate and produce immunoglobulins of all other types, and are responsible for initiating B-cell responses by activating naive B cells to proliferate and secrete IgM. The principal membrane- bound effector molecule expressed by TH2 cells is CD40 ligand, which binds to CD40 on the B cell and induces B-cell isotype switch, humoral immune response. • The TH17 subset is primarily produce IL-17 that involved in recruiting neutrophils and macrophages to site of infection, inducing inflammation and cause some autoimmune diseases. Helper T cells subtypes
- 53. Summary
- 54. Cloning ensures abundance of specfic response
- 55. T reg • Regulatory T cells are generated mainly by self antigen recognition in the thymus (central) and by recognition of self and foreign antigens in peripheral lymphoid organs (peripheral) • The generation of some regulatory T cells requires the cytokine TGF-β+IL2 Treg nTreg pTreg iTreg
- 56. Treg Function
- 57. Th17/Treg axis is a complex axis of contradicting powers Pawel Muranski et al 2013
- 58. Transcription factor as master regulators or Not? Sara Omenetti et al, 2016
- 59. Open questions • How to ensure that when we target, we actually regulate one phenotype and leave the others ? • How to convert between different cell types in natural way • Role of microbiota • Role of the gut brain axis • Cancer immunotherapy : which cell to target ?
Editor's Notes
- #20: cor·tex /‘kɔrtɛks/ noun, plural cor·ti·ces /ˈkɔrtəˌsiz/ Show Spelled . 1. Anatomy, Zoology . a. the outer region of an organ or structure, as the outer portion of the kidney. b. the cerebral cortex. me·dul·la /mə‘dʌlə/ noun, plural me·dul·las, me·dul·lae /məˈdʌli/ Show Spelled . 1. Anatomy . a. the marrow of the bones. b. the soft, marrowlike center of an organ, as the kidney or adrenal gland. c. medulla oblongata.