III Week 3

Where in lymphoid tissue would Ags end up in if they - Entered through wound in skin.... - Entered through gut..... - Were in the blood stream....

1) Skin --> ECF --> lymphatic vessels in dermis --> go to regional lymph nodes 2) Gut --> absorbed by endocytosis by cells lining MALT (Peyer's patches) --> presented to lymphocytes 3) Blood stream --> enter spleen via splenic arteries

Funtion of Treg cell

Inhibit T cell activation by interacting with D.Cs in secondary lymphoid tissue

Role of IL-2 in T cells

• Once the T cell gets all the signals it needs, it starts to proliferate. It can release cytokines (such as IL-2) which can drive its own proliferation

What are the main roles of TH17 cells?

• One of their key roles is to drive inflammation, particularly the recruitment of neutrophils to the site of inflammation

What initiates signalling into the T cell?

• Signal transduction through signaling proteins associated with TCR is required to activate the T cell • Ag binding leads to phosphorylation of signaling proteins, cascade of protein: protein interactions • Increased expression of certain genes e.g. cell cycle, differentiation, cytokines • CD3 is a T cell marker, which is intimately associated with the TCR • ITAm are signaling molecules

What genes do the TCR alpha and beta loci contain?

• TCR locus - V (variable) & J (joining) gene segments (V and J) & C (constant) gene • TCR locus contains V, J & D (diversity) gene segments (V, J, D& 2 constant genes • Same enzymes involved as the BCR gene rearrangement - RAG involved in recombining the different gene fragments

Where does T cell activation occur and what are its 4 steps?

• If it has a better fit with MHC I, CD4 will be down regulated, and if it has a better fit with MHC II, CD8 will be down-regulated - what it becomes depends on the APC

What leads to T cell inactivation (anergy)?

Ag Recognition in Absence of Co-stimulation leads to T cell Inactivation (anergy) or Deletion = Peripheral Tolerance • TCR activation without CD28/B7 co-stimulation leads to T cell anergy (unresponsiveness) • Anergic cell is stuck, being inactive - these cells are not very helpful in terms of effector function - Very often, cells don't have co-stimulation because it prevents them from becoming self-reactive

What are the different fate-specifying cytokines for the different kinds of CD4 T cells?

Cytokines are the principal determinants of alternative programs of CD4 T-cell effector differentiation. Antigen-presenting cells, principally dendritic cells, as well as other innate immune cells can provide various cytokines that induce the development of naive CD4 T cells into distinct subsets. The environmental conditions, such as the exposure to various pathogens, determine which cytokines innate sensor cells will produce. TH1 cells differentiate in response to sequential IFN- and IL-12 signaling, whereas TH2 cells differentiate in response to IL-4. IL-6 produced by dendritic cells acts with transforming growth factor- (TGF- ) to induce differentiation of TH17 cells, which upregulate expression of the IL-23 receptor and become responsive to IL-23. TFH cells also require IL-6 for their development, although it is not currently understood what additional signals might induce their differentiation from naive precursors. When pathogens are absent, the presence of TGF- and IL-2, and the lack of IL-6, favor the development of induced Treg cells.

Explain the following image...

Subsets of CD4 effector T cells are specialized to provide help to different target cells for the eradication of different classes of pathogens. Unlike CD8 T cells, which act directly on infected target cells to eliminate pathogens, CD4 T cells typically enhance the effector functions of other cells that eradicate pathogens—whether cells of the innate immune system, or, in the case of TFH cells, antigen-specific B cells. TH1 cells (first panels) produce cytokines, such as IFN- , which activate macrophages, enabling them to destroy intracellular microorganisms more efficiently. TH2 cells (second panels) produce cytokines that recruit and activate eosinophils (IL-5) and mast cells and basophils (IL-4), and promote enhanced barrier immunity at mucosal surfaces (IL-13) to eradicate helminths. TH17 cells (third panels) secrete IL-17-family cytokines that induce local epithelial and stromal cells to produce chemokines that recruit neutrophils to sites of infection. TH17 cells also produce IL-22, which along with IL-17 can activate epithelial cells at the barrier site to produce antimicrobial peptides that kill bacteria. TFH cells (fourth panels) form cognate interactions with naive B cells through linked recognition of antigen and traffic to B-cell follicles, where they promote the germinal center response. TFH cells produce cytokines characteristic of other subsets and participate in type 1, 2, and 3 responses that are recruited against different types of pathogens. TFH cells producing IFN- activate B cells to produce strongly opsonizing antibodies belonging to certain IgG subclasses (IgG1 and IgG3 in humans, and their homologs, IgG2a and IgG2b, in the mouse) in type 1 responses. Those TFH cells producing IL-4 drive B cells to differentiate and produce immunoglobulin IgE, which arms mast cells and basophils for granule release in type 2 responses. TFH cells that produce IL-17 appear to be important for generating opsonizing antibodies directed against extracellular pathogens in the context of type 3/TH17 immunity. Regulatory T cells (right panels) generally suppress T-cell and innate immune cell activity and help prevent the development of autoimmunity during immune responses.

Where are thymocytes at different developmental stages found in the thymus?

The earliest precursor thymocytes enter the thymus from the bloodstream via venules near the cortico-medullary junction. Ligands that interact with the receptor Notch1 are expressed in the thymus and act on the immigrant cells to commit them to the T-cell lineage. As these cells differentiate through the early CD4-CD8- double-negative (DN) stages, they migrate through the cortico-medullary junction and to the outer cortex. DN3 cells reside near the subcapsular region, where they undergo proliferation. As the progenitor matures further to the CD4+CD8+ double positive stage, it migrates back through the cortex. Finally, the medulla contains only mature single-positive T cells, which eventually leave the thymus.

Describe the rearrangement of the alpha and beta chain genes...

alpha Chain • VJ alpha gene segment rearranges to a J alpha segment • Transcription & splicing to C alpha generates mRNA that is translated to yield the TCR alpha-chain • More like the light chain as there are no D gene fragments Beta Chain • VDJ Beta exon is transcribed & spliced to C Beta • mRNA translated to yield TCR beta chain • have V, D and J gene fragments along with the constant genes • The TCR alpha- and beta-chain genes are composed of discrete segments that are joined by somatic recombination during development of the T cell. Functional alpha - and beta-chain genes are generated in the same way that complete immunoglobulin genes are created. For the alpha chain (upper part of figure), a V gene segment rearranges to a J-alpha gene segment to create a functional V-region exon. Transcription and splicing of the VJ exon to C generates the mRNA that is translated to yield the T-cell receptor alpha-chain protein. For the beta-chain (lower part of figure), like the immunoglobulin heavy chain, the variable domain is encoded in three gene segments, V-beta, D-beta , and J-beta . Rearrangement of these gene segments generates a functional VDJ-beta V-region exon that is transcribed and spliced to join to Cbeta ; the resulting mRNA is translated to yield the T-cell receptor chain. The alpha and beta chains pair soon after their synthesis to yield the alpha:beta T-cell receptor heterodimer. Not all J gene segments are shown, and the leader sequences preceding each V gene segment are omitted for simplicity.

Can TCRs recognise peptides on their own? What is the exception to the rule?

• APC uptakes the Ag and chops it into peptides (up to 20 amino acids long) • APC can express MHC classes I and II at the same time - can simultaneously interact with different types of T cells • TCRs bind short peptide fragments derived from intact protein (contrast from BCR/Ig) • TCR recognizes Ag in form of complex of peptide bound to an Major Histocompatibility Complex (MHC) molecule on Antigen Presenting Cells (APC) • TCR doesn't recognize peptides not bound to MHC • Exception! - Superantigens - can bind TCR/MHC without processing to activate T cells ϖ Often produced by things like streptococcous which can cause toxic shock syndrome ϖ Can cause the T cell to be activated, no matter what that T cell is supposed to recognize ϖ Get lots of T cells being activated simultaneously

How are Naive T cells activated and stimulated to differentiate? What molecules are involved in the different signals required?

• APCs deliver 3 signals for proliferation & differentiation of naïve T cells • Ag:MHC complex binding to the T cell receptor (TCR) on naïve T cells transmits a signal to T cell that antigen has been recognized (signal 1) - Kick starts the T cell to think about proliferating - Stimulation signal • Signal 1 alone not adequate for the activation of T cell • Activation of naïve T cells requires 2nd signal (signal 2) known as a co-stimulator signal delivered by the same APC - Without this signal, the T cell wont become fully activated • CD28 dependent co-stimulation triggers T cells to enter cell cycle & induces synthesis of IL-2 & its receptor Il-2R • IL-2 drives T cell proliferation - clonal expansion • Signal 3 (cytokines) drives differentiation - CD4 needs cytokine signals - these cytokines tell the T cell whether to become TH1, TH2, TH17 or Treg

What is the aim of T cell maturation and what are the main processes involved?

• Aim - to generate lots of different T cell clones with different TCRs that recognize many different foreign peptides & to eliminate T cells that react with self peptides i.e. autoreactive clones • Rearrangement of genes encoding TCRs - Outcome is that each T cell receptor has a different sequence, due to different choice of gene fragments and the errors introduced during the cutting and joining process • Thymic selection - positive and negative - Want to select the ones we want and get rid of the ones we don't want • Mature naïve T cells exit thymus into peripheral lymphoid organs

What are the different types of effector T cells, their main functions in the adaptive immune response and the pathogens they target?

• CD4 T cells - naïve CD4 differentiate into any one of a number of different effector cells with different function defined largely based on cytokines they produce • CD8 T cells - naïve CD8 differentiate into cytotoxic cells - kill target cells

What does CD8 T cell differentiation require?

• CD8 responses usually require CD4 help but can occur independently if initial stimulus is strong enough • CD4 T cells enhance CD8 T cell responses • CD4 T cells important for CD8 T cells memory responses • T helper cell can provide a little bit of IL-2 to help its differentiation

What does CD8 T cell differentiation require?

• CD8 responses usually require CD4 help but can occur independently if initial stimulus is strong enough • TH1 cell produce IFN-gamma which is a fate specifying cytokine of CTLs • CD4 T cells important for CD8 T cells memory responses • T helper cell can provide a little bit of IL-2 to help its differentiation

How do CTLs cause cytotoxicity?

• CTLs induce target cells to undergo apoptosis • Cytotoxic effector molecules that trigger apoptosis are contained in granules • Engagement of TCR with MHC class I: peptide on target cell induces release of perforin, granzymes, granulysin - Toxic molecules that induce apoptosis • Intracellular targets of granule contents include caspases, nuclear DNA, mitochondrial proteins • Release of mitochondrial cytochrome c, induces DNA fragmentation > apoptosis > apoptotic cells cleared by phagocytes • Other pathways of apoptosis (death receptors e.g. Fas)

What cells does the thymus contain and what happens within the thymus? Where does it sit?

• Common lymphoid progenitor cells start to populate the thymus early in embryogenesis fully developed at birth, after puberty involution • Site where T cells are educated to self before export to periphery • Epithelial cells, dendritic cells/macrophages present self peptide to maturing T cell (positive & negative selection) • Majority of lymphocytes entering thymus do NOT leave (98% destroyed) • Thymus sits just above the heart • Quite structured - has different lobes and segments - Within each segment, have a cortex and a medulla - maturing T cells are found throughout these - As they mature, they move through the cortex and are interacting with stromal cells and various types of APCs - They get to a point where they are expressing T-cell relevant molecules (such as CD3, CD4, CD8) - they then move to the medulla to undergo selection and travel from here into the blood, into the secondary lymphoid tissues • A lot of the T cells get deleted - want the T cells to have a functional TCR that doesn't bind to self-antigen

Summarise T cell Ontogeny

• Developing T cells pass through distinct phases • Enter thymus as CD3-CD4-CD8-TCR- (double negative, (DN) thymocytes) • V(D)J gene rearrangement • Beta chain selection • Positive selection • Leave thymus as naïve but mature CD4+ or CD8+ T cells • T cells enter peripheral lymphoid organs, MALT, spleen DON'T WORRY ABOUT THE MOLECULES AT THE BOTTOM • Beta chain recombination occurs first, then the alpha chain • As the T cell goes through DN stages, it interacts with other cells such as cortical epithelial cells and there is testing for self-reactivity • There is no membrane to cross over as it matures, it just goes deeper into the tissue (medulla)

What are the main functions of TH2 cells?

• For most Ags B cells need help from T helper cell to start making Ab • Helpter T cells activate B cells that recognize the same Ag • Interactions between MHC class/peptide on B cell & TCR on CD4 T cell + CD40:CD40L • IL-4 secreted by Th2 cell towards B cell • Ab secretion, class switching to certain isotypes

Describe the structure of the TCR...

• Heterodimer - 2 transmembrane proteins • TCR chains consist of variable (V) amino terminal region & constant (C) region • Gene locus for each chain • Mechanics of gene rearrangement is similar in T and B cells, similar process and enzymes involved • T cell receptor gene rearrangement takes place in the thymus • T cell maturation - how you end up with the alpha and beta chains

What are the effector functions of the CD8 T cell?

• Limited - killing is what they do • Activated CD8 T cells differentiate in cytotoxic T cells (CTLs) with help from Th1 cells i.e. IFN • CTLs kill target cells that display peptides of cytosolic pathogen on their surface bound to MHC class I molecules • CTLs induce apoptosis in target cells • Important in defence against intracellular pathogens (also tmours) • Naïve CD8 T cells > interact with dendritic cells > differentiate into CTL > killing of target cell expressing its peptide+MHC class I • CTLs are selective - only cells displaying their specific Ag are killed NOTE: MHCI found on all nucleated cells, decreased some infections, tumours • CTLs also release cytokines - IFN inhibits viral replication, directly, activates macrophages - TNF recruitment of other inflammatory cells, promote target cell killing

How does T cell activation occur in the Lymph nodes?

• Naïve T cells migrate to lymphoid organs via the blood • Lymphocytes leave the blood & enter the LNs via the high endothelial venules • Exit through the efferent lymphatics • Ag is delivered to the APC, which are in the same spot as the naïve T cells - APCs grab the antigen from the lymph node and present it OR the activated dendritic cells come in and activate the T cells - If they are helper T cells, they can move in and help the B cells • Activation of the T cells only when they come across their specific Ag • The activated T cells then migrate to where they are needed (e.g. CD8 cells may travel to the skin where there is an infection) • Same thing happens in the spleen, where the Ag comes in via blood, not the lymph

Where do T cells mature and where are they activated?

• T cells develop in thymus from lymphoid progenitor cells that leave the BM and go to the thymus • They get initial signals to start maturation by the stromal cells • They start to rearrange their genes for the alpha and beta chains • They are selected - those who react to self-molecules are killed (via apoptosis) • They then leave the thymus, go into the blood and enter the secondary lymphoid tissues - where they encounter their Ag - They encounter their Ag presented by an APC - present peptide Ag • T cell becomes activated and may differentiate further - Naïve CD8 T cell can become a CTL - Naïve CD4 T cell can become a type of helper cell

When do T cells differentiate into effector or memory cells?

• T cells differentiate into effector or memory cells after Ag encounter • Effector functions of T cells are determined by the array of effector molecules they produce • Effector molecules e.g. cytotoxins or cytokines • CD8 T cell once activated, doesn't have much of a choice - will become a CTL or a memory cell • CD4 T cell has many choices of what to become - all different subsets have different roles in the immune response

How does T cell activation and differentiation occur following Ag exposure?

• T cells exit the thymus as naïve CD4 T cells or CD8 T cells • Antigen recognition - MHC molecules on APCs present peptide to naïve T cells = antigen presentation • T cell differentiation & proliferation • Effector function - e.g. CD4 T cells help B cells, activate macrophages; CD8T cells become CTLs, kill target cells • Occurs in peripheral tissues - lymphoid organs & other tissues • APC is presenting Ag via MHC to the T cell which is recognized by the TCR - In this image, it is a CD8 T cell as the antigen is being presented by MHC class I - CD4 = MHC class 1 - CD8 = MHC class 2 • There are a number of other signals the T cell gets from the APC - B7 on APC and CD28 on T cell - T cell requires additional signals from the APC - CD4 T cells also need a third signal to determine what type of helper cell to become • Once the T cell gets all the signals it needs, it starts to proliferate. It can release cytokines (such as IL-2) which can drive its own proliferation

How is Ag presented to CD4 and CD8 T cells? What molecules are involved?

• T cells see & respond to Ag only in complex with MHC • CD4 T cells respond to Ag with MHC class II • CD8 T cells respond to Ag with MHC class I • How do CD4 or CD8 T cells know? - CD4 molecule has specific binding site for MHC class II & CD8 molecule has specific binding sites for MHC class I - CD8 molecule only fits with MHC class I - doesn't have a shape to fit with MHC class II • B7 = CD80/86 on the APC interacts with CD28 on the CD8 cell - these molecules provide the second signal to the T cell for activation - essential! • CD4 gets more interaction with the APC than the CD8 - there is CD40 interacting with CD40L

Where does T cell maturation and thymic selection occur? What is positive and negative selection and how do they occur?

• This happens throughout the thymic lobes (not just in one spot) • DN cells proliferate & differentiate into double positive (DP) thymocytes • Unless they recognize pMHCI (engaging CD8) or pMHCII (engaging CD4) expressed by cortical epithelial cells - they die • This is called positive selection - Signal to survive • Thymocytes surviving positive selection express only CD4 or only CD8 in addition to CD3 & TCR • Thymocytes move into medualla • Medullary thymocytes that interact very strongly with self peptide on MHC1 or pMHCII on APCs undergo apoptosis • This is called negative selection - Signal to die • Mature single positive (SP) thymocytes enter venules & exit thymus as mature naïve T cells • There are other mechanisms to stop self-reactive cells that bypass these processes, and if they fail, we get autoimmune diseases

What is immunological memory and what is it mediated by?

• This is still all theoretical • Most activated T (and B) cells become effector cells • Some then become memory cells • Memory cells are long-lived cells & persist at higher levels than naïve lymphocytes (100-1000 fold above initial frequency) • Respond rapidly to Ag challenge compared to naïve lymphocytes • Memory cells can rapidly differentiate into effector cells after Ag exposure • Memory cells, vaccines, secondary infection - We try to get a large number of memory cells in the body when we administer vaccines • Once a T cell has seen Ag, some will become memory and some will become effector