Module 8

What causes T cell to proliferate?

-Binding of IL2 to IL2 receptor -naïve T cells express moderate/low-affinity IL-2R, doesn't do much -activation of T cells leads to expression of IL-2Rα and a high-affinity IL-2R If they make alpha chain, it is high affinity, leading to proliferation. Alpha chain is CD25.

Recall from Chapter 3

-During an innate response, NK cells secrete IFN-g (cytokine) that activate macrophages to be better phagocytic cells, making more lysosomes, lysogenic enzymes, and MHCs. NK cells provide mechanism to activate dendritic cells

Fate of T cells in the LN

-T cells that do not encounter antigenic peptide-MHC can continue to migrate to additional LNs and eventually back into the blood and to different LNs -T cells that do encounter antigen-MHC will proliferate, differentiate into effector cells, and either remain in the LN or migrate

Organization

1. Role of dendritic cells (DCs) in T cell activation (basically the only one that can activate a naive T cell) 2. Homing of DCs and T cells to the lymph node 3. T cell-DC interactions (forming a synapse called the SMAC) 4. TCR signaling for activation 5. T-cell differentiation and role of different T-cell subsets. Effector cells: a. CD8 b. CD4 6. Importance of CTLA-4 Maybe some other stuff

Effector T-cell functions are mediated by cytokines and cytotoxins

Effector functions p 2 with CD8 CD8 carry out effect primarily by secreting cytotoxins (perforin, granulysin, serglycin and granzyme). Poke holes into virally affected cells, causing them to die. CD4 functions are carried out by cytokines they secrete.

Another way to look at this T cell receptor signalling cascade

IP3 works on calcium stores causing flux, activates NFAT. IL-2 and IL-2Ra expressed by T cell Goes through the PLCg activation, pip -> IP3, DAG. End result is NfKB, NFAT, and AF-1 Enough signal 2 leads to enough NFkB so it can proliferate and differentiate. Leads to production of alpha chain and IL2

SMAC or Immunologic Synapse

LFA-1: an integrin; binds ICAM on APC Confocal microscopy

Visualization of CTL killing

Pulsed Picture: bunch of macrophages that express MHC that T cells have receptors for.

RH17

ROR-gt Leave lymph node and go out to site of infection, enhance neutrophil response, cause excess production of CXCL8, enhancing recruitment of neutrophils Other functions later in the powerpoint

TH1

T-bet Activate macrophages Leave lymph node and migrate to site of infection Other functions later in the powerpoint

Activation of CD4+ T Cells p 3

Transcription factor- NFkappaB Produced in 2 ways DAG pathway-> PKCt -> removal of iKB to move to nucleus Augmented by CD28 signal T cell w/o signal 2 doesn't activate enough NfKappaB to proliferate. Need tons of NFKB. Byproducts of transcription of NFKB is BCLXL- antiapoptotic gene, blocks apoptosis. Mitochondrial membrane protein. Blocks reactive oxygen species production that leads to caspase activation. Allows T cells to survive long term.

Treg

suppress activation of other effector T cells often autoreactive, made in thymus and released but can also develop in lymph node where they have the same characteristic, secreting TGF-B and IL10 onto surrounding T cells, suppressing activation of surrounding T cells Role in tolerance (peripheral) Other functions later in the powerpoint

What's next 2:

¡Cytokines change the patterns of gene expression in the cells targeted by effector T cells ¡Cytotoxic CD8 T cells are selective and serial killers of target cells at sites of infection ¡Cytotoxic T cells kill their target cells by inducing apoptosis ¡Effector TH1 CD4 cells induce macrophage activation ¡TFH cells, and the naive B cells that they help, recognize different epitopes of the same antigen ¡Activated T cells express CTLA-4, which slows T cell activation as the adaptive response wanes. Slows down immune response.

Previously activated T-cells do not proliferate due to CTLA-4 expression

-Counterpart to CD28 -signal 2 comes from B7 interacting with CD28 on the T cell. -Once T-cell is activated, effector T cells decrease level of CD28 on the surface and it expresses CTLA4 (stronger interaction with B7 molecules, family member of CD28) -Signal does nothing in the T cell (?) CD28 doesn't bind anymore -Activated T cells that are out recognizing MHC and peptide on antigen presenting cells that have B7, CTLA4 bindsto B7 in place of CD28 ¡CTLA4 signaling limits proliferation and at high levels, causes anergy -Cell cycle arrest. Can't reactivate previously activated T cell. Important with activated macrophages at the site of infection, for autoreactive CD8 T cells or 2 populations of cytotoxic T cells, one recognizes peptide and other is a memory cell that isn't needed. Memory cell would use up resources. CTLA4 prevents this. Similar to PD1 protein in cancer -Effector T cells, we don't want them to start proliferating randomly in the periphery. Highly regulated function. -Mechanism to autoregulate -No signal 2 needed

Activation of adaptive immunity requires activation of T-cells

-Dendritic cells carry antigens from sites of infection to secondary lymphoid tissues. This is what activates T cells. -Dendritic cells are adept and versatile at processing antigens from pathogens -Dendritic cells express MHC1 and MHC2 -Not a powerhouse phagocytic cell like macrophages are -Survey the periphery and endocytosis -Micro and macroendocytosis -Found in those tissues that are in contact with the external environment Skin - Langerhans cells (self-renewing) Nose, lungs, stomach, intestines Blood - immature state

The Granuloma

-Macrophage is phagocytosing pathogens but the T cells are only providing one type of help. -The interferon gamma receptors that the macrophages produce are upregulated when they get CD40 signal from CD40 ligand. -Intravesicular pathogens evade killing -Losing signal- not as responsive to interferon gamma from the T helper cells, not as good at phagocytosing -Depending on what bacterium is. If it is one that likes to live in vesicles, it evades lysosomes by budding off in some sort of side vesicles that don't get targeted by the lysosome and the bacteria cam live inside. ¡Mycobacterium and Leishmania (pushes its way out of vesicle before it fuses with lysosome and lives in cytosol) Chronically infected macrophages will build up where the pathogen is, and sometimes they fuse together with multiple nuclei inside. T helper cells are outside but can't help because they don't have the proper signals (ex: CD40 ligand). ¡Giant cells form - fusion of infected macrophages ¡TH1 cells surround macrophages ¡sometimes these get large and are cut off from blood supply and the center cells die. Can result in cysts that are empty on the inside. Can destroy tissue around it. Inside of granuloma is filled with macrophages and dying macrophages and bacteria since the macrophages can't kill.

Peripheral T cell Tolerance

-Negative selection isn't perfect, can bind to MHC and self peptide with enough strength to get activated with signal 1 and 2 -Activated antigen presenting cell presents lots of foreign peptide and B7 -Need for both signal leads to peripheral tolerance ¡Signal #1 WITHOUT signal #2 (maybe presenting self peptide, no B7) leads to anergy and T-cell tolerance - Requirement of ADJUVANT. No longer responsive through T cell receptor. ¡Signal #2 WITHOUT signal #1 leads to nothing! May still bump up against dendritic cells, but if they don't recognize peptide and MHC, they don't get signal 1. ¡Anergic T cells do not respond even if signal #1 and #2 are present Anergic- shuts down IL2 and IL2Ra protein, can't proliferate, die within several days Primary peripheral tolerance mechanism Autoreactive T cells that get out recognize MHC and self peptide on cells, but if they recognize it on cells w/o B7 then they don't get signal 2. Turns anergic, but could come off and bump into a dendritic cell expressing B7, it won't do anything since it is always anergic.

Recap 2:

-T cells bind to MHC and get signal 2, driving proliferation and differentiation, production of IL2 and IL2 receptor ¡Proliferation and differentiation of activated näive T cells are driven by the cytokine Interleukin-2 ¡Antigen recognition in the absence of co-stimulation leads to a state of T-cell anergy (w/o signal 2) ¡The cytokine environment determines which differentiation pathway a näive T cell takes (fate, effector cell type) ¡Näive CD8 T cells require stronger activation (CD2) than naive CD4 T cells ¡Effector T-cell functions are mediated by cytokines and cytotoxins

Macrophages in secondary lymphoid tissue

-macrophages and B cells (to a lesser extent) can express B7, all over in lymph node ¡Found throughout the LN ¡Function to clear debris that arrives from the lymph ¡Function to clear lymphocytes that die in the tissue -Activated because it is phagocytosing pathogen and probably receiving interferon gamma signals. ¡Activated Macrophages can upregulate B7 and MHC expression BUT not enough to activate naïve CD8+ T cells (signal 2) since they need a lot of B7. -Macrophages in the LN aren't from the site of infection, came in from HEV. If pathogen comes in from lymphatic then macrophages can destroy it. CD4 outnumber CD8, more likely they provide signal 2 for CD4 T cells.

T-cell activation requires co-stimulation

-¡Remember, the TCR cannot signal alone. --Signalling cascade from t cell is signal 1, not enough by itself. ¡Additional molecules include CD3/z and either CD4 or CD8 -Needs signal for CD28 with B7 ¡CD28 is required for activation ¡CD28 binds B7.1/B7.2 (CD80/CD86) on the DC. Form a dimer causing the second signal . ¡Once T-cell is activated, it expresses CTLA4 (stronger interaction with B7 molecules, talked more about below) ¡CTLA4 signaling limits proliferation -Need both signal 1 and signal 2 Signal 2 Requires dendritic cell to express B7. If T cell binds to B cell or macrophage, they express low/no levels of B7. Dendritic cell expresses a lot.

Co-Stimulatory Molecules are Required for T cell Activation!

1. TCR - MHC + antigen and CD4/8 - MHC II/I 2. CD28 - B7 These interactions are required for the correct signal to be sent for T cell proliferation and differentiation into effector cells!!!

APCs of the lymph nodes

1.B cells •Primarily in the germinal centers of the cortex -Can express B7 2.Macrophages •Resident •Lymph node location: cortex and medulla -best because they are migrating from infection and express a lot of B7 3.Dendritic cells (hematopoietic origin) •Highly migratory •Lymph node location: T-cell regions of the cortex -Can express B7 -Most important T cell activator for naive T cells recognizing MHC and peptide for the first time because they need signal 2

DCs are adept at processing and presenting antigen - BUT not same as Macrophages

Activation through signalling through tlr Downstream function- activation of dendritic cell Adept at dealing with pathogen/sampling debri with a bunch of different mechanisms -Endocytose w/ receptor -Endocytose w/o receptor (macropinocytosis) with bacteria and viruses - Viruses in cell Dendritic cells can sample debri from other cells. Virus can infect dendritic cell. Virulence factors downregulate the dendritic cell. Can dump debri outside of cell that can be present on MHC1 to activate CD8 T cells. (Or MHC2) Good at cross-presentation: Phagocytose = MHC1 Viral protein stick on MHC2 Different than macrophages, which are not as good at cross presentation but are good at phagocytosing/destroying and can enhance degree of destruction. Dendritic cells are active from the start.

Activation of naive CD4 T cells gives rise to effector CD4 T cells with distinctive helper functions

After it gets signal 1 and 2 and is proliferating Due to NFKB, NFAT, AP1 During proliferation, express different transcription factors that leads to differentiation to effector. Differentiate based on the cytokines around them, phenotypes. T-bet, ROR-gT, GATA3, BCL, FoxP3. Help with transcription factors. 5 helper T cell subsets Express and secrete the same cytokines that activate them Need to know different effector functions TH1, TH17, TH2, TFH, Treg (regulatory cells) As they differentiate (same time as proliferating), they are secreting at least 1 cytokine involved in differentiation (Tfh is bad at this) Function CD8 have one effector T cell type, killer t cells, not dependent on the cytokines.

Activation of naive CD4 T cells gives rise to effector CD4 T cells with distinctive helper functions (part 2)

Bind to cytokine receptors

Remember Group 3 Lab Case??

CD40 ligand deficiency If in those patients, T cells still expressed CD25 Common marker for activation of T cells once activated and proliferated

CTLs kill target cells in a successive fashion

CD8 cytotoxic T cells activated in the same way, except they need more B7. Effector function is independent of cytokines, which is to bind to MHC and peptide. CTLs can "pick-out" the infected cells from the healthy cells and can successively kill adjacent infected cells Recognizes infected cell, secretes toxins onto the cell that start to kill it, then moves onto neighboring cell, etc Determines infected cell from noninfected cell through multiple mechanisms. Primary mechanism is presentation of viral peptide on MHC1 and the t cell. Once a T cell is activated, it never needs B7 ever again. Once activated, it only needs signal 1.

Cytokines and transmembrane proteins produced by TH1 cells

Effector cells carry out effector function based on cytokines they secrete. Primary role of TH1 cell (though they can have an effect on B cells as they are being made in the lymph node) is to migrate out of the lymph node and go back to the tissue and release a bunch of cytokines This is a list of the cytokines and other things that TH1 cells secrete (and functional role) Activate macrophages to make them better at engulfing and destroying pathogen Can cause chronically infected macrophages to basically die and release their contents Can produce IL2 to produce more Th1 cells. In the lymph node when the T cell is first getting activated, makes cytokine to drive other T cells to become TH1 cells. Secretes stimulating factor, granulocyte monocyte stimulating factor. Growth factor in the bone marrow that drives differentiation progenitor cells. Drive precursors to become monocytes and macrophages. Make more macrophages and help increase ability of monocytes to come out of the blood and migrate into the tissue and become macrophages. Help produce chemotactic factors that help draw these in. Loosen the vasculature and they produce the chemotactic signals for the monocytes to go back into the tissue. Important if the pathogen isn't destroyed by innate response, calls in more help at the site of the infection.

TH2 IL-4 drives TH2 skewing

Example: TH2 cells that drive IL4 can drive production of more TH2 cells. Example of T cell that recognizes MHC and peptide on a dendritic cell in the lymph node and starts to proliferate. If IL4 is around, it drives it to differentiate into a TH2 cell to express the g3 transcription factor to make more IL4 and IL5, which can drive more T cells to do this. Two different receptors that IL4 can bind to (both chains ARe specific for IL4) Gamma C (common gamma chain)- transmembrane protein that is utilized in formation of cytokine receptors of a whole bunch of different types Protein recognizes IL4 and the other that comes along with it. Other side: one that recognizes IL4 and one that recognizes IL13, which are in the same family of cytokines and have similar effects. STAT6 gets phosphorylated and forms a homodimer, and then that transcription factor goes into the nucleus and you can see it starts to initiate transcription of GATA 3. Cell receiving this IL4 receptor makes gata 3. This skews developing T cell to become a TH2 cell. Other functions of IL4 secreted by TH2 cells- drive B cells to class switch to IgE. Transcription factors that are produced in response to IL4 in the B cell open up class switching site to IgE to allow class switching to become an IgE immunoglobulin. Th2 also has function in the brain. Ligation of receptor for IL4 leads to specific type of STAT dimer that leads to some functional characteristics of the TH2 derived cytokine.

TFH

Go to follicle and initiate germinal center response. Responsible for activating B cells and driving them into germinal center where they proliferate, go through somatic hypermutation and class switch Maturation of antibody response Key to high affinity class switched antibodies Other functions later in the powerpoint

Macrophage Phenotypes are also regulated by cytokines

Have different phenotypes and different effector functions. Can change effector functions based on cytokines that are present. Unlike T helper cells ( who, once they differentiate to be a certain type of effector cell, they stay that effector cell for the rest of their life span), macrophages don't. M1 macrophage- host defense macrophage, one that is activated by interferon gamma, binds to pathogens through its pathogen recognition or pattern recognition receptors and phagocytoses and makes proinflammatory cytokines (IL12, IL, IL1B, TANFa) and chemokines (IL8/CXCL8) that recruit neutrophils into the site of the infection, and other chemokines that recruit more monocytes into the infection. As pathogenic load starts to decrease and macrophage eliminates the pathogen, and as dendritic cells start taking the pathogen out of the site into the lymph nodes and initiating an adaptive response, macrophages start to change their phenotype to this tissue repair macrophage M2a macrophage- driving force is cytokine IL4 (which comes from TH2 cells, which come from the lymph nodes into the site of the infection, secreting IL4, IL13). Macrophages change phenotype from proinflammatory to one that tries to repair/secretes factors to help repair the tissue where there is an inflammatory response taking place. There is a lot of damage and reactive oxygen species and other cell death. Secrete growth factors and MMP (matrix matala proteases) which chop up the extracellular matrix. Growth factors, like basic fibroblast growth factor, lead to production of more fibroblasts, which lays down new extracellular matrix. Platelet derived growth factor (PDGF), vascular and endothelial growth factor rebuild vasculature in the damaged site. Usually not recruited monocytes (though they can be). Makes more IL10, which is an anti-inflammatory cytokine. Shuts down activation of macrophages and their production of pro-inflammatory cytokines, inhibiting other pathways that activate more M2. Resolution phase- another phenotype. IN the presence of IL10, produced by M2 cells, certain T helper cells, and regulatory T cells. IL10 can drive M2 to become resolution macrophage. Secretes more IL10 and transforming growth factor beta, which is important in other cell proliferation events. Finalizes remodeling of the tissue. Can be reactivated to M1 if it gets interferon gamma Macrophages are not always killers.

Cytotoxic CD8 T cells are selective and serial killers of target cells at sites of infection

How do CTLs target their lytic granules to the infected cell? Use a redistribution of the cytoskeleton T cell binds using cell adhesion molecules, allowing for T cell receptors to can the MHC and peptides. Lytic granules on the inside. Microtubule organizing center builds microtubules to help form the internal workings of the process. When T cell recognizes the MHC and peptide and starts getting signals back and forth between these cells, it causes T cell to reorganize intracellular machinery so golgi and vesicles (move from ER to golgi) are pointed in direction of contact/synapse. Allows for migration of vesicles that have lytic granules that the cytotoxic T cells use to the surface that is adjacent to the target cell. Lytic granules are dumped on the target all around outside of contact. Form a ring around contact point (synapse) that helps hold the lytic granules that are dumped inside the synapse so they target the cell and not neighboring cells. Able to pinpoint specific cell, not hit the other cells, and dump the granules. Then it tests the neighboring cells.

T-cell "recognition" results in conformational change in LFA-1

If it recognizes peptide and the CD4 or CD8, leads to a signal causing LFA1 to change it's 3d structure to really clamp down, slowing migration in the tissue. This is called cognate pair: T cell and T cell receptor recognize MHC and peptide on another cell. Stop, interact, hold together for a while. Can hold together for up to a week. T cell signals through its proteins to cause T cell to differentiate and proliferate (2-3 times a day, each cell forming cognate pair, leading to thousands of T cells)

TFH cells, and the naive B cells that they help, can recognize different epitopes of the same antigen

Linked recognition CD4, function in the lymph nodes or in secondary lymphoid tissue. Drive B cell to the follicle and allow it to class switch/hypermutate. Interacts with MHC2 on surface of the B cell. Provides second signal through CD40 because it expresses CD40 ligand and it secretes cytokines. CD40 is essential. Linked recognition: B cell doesn't necessarily have to recognize proteins, it can recognize carbohydrates, lipids, carbohydrates on proteins etc. Picture shows B cell receptor binding to blue substance with specificity. When the blue protein is phagocytosed and broken up, other peptides are made. In picture, MHC picks up red portion of the antigen. Takes it to the surface, and if there is a T cell that has a receptor that recognizes that MHC and red peptide, it will respond to it. B cell technically recognized something different than what the T cell did. T cells have ability to recognize specific peptide independent of the B cell. Since MHC only present peptides, this link recognition is beneficial.

Cytokine signaling involves JAK receptors and STAT transcription factors

Made up of 2 subunits. Usually one that is specific and one that can bind to multiple. Cytokines change the patterns of gene expression in the cells targeted by effector T cells Bind to receptors that are either homo or heterodimers Cytoplasmic domains are associated with JAKS (kinase family). When cytokine binds, the kinase phosphorylates the cytoplasmic domain of receptor and phosphorylate each other, leading to a binding domain for STAT proteins (family, signal transduction associated transcription factor). Associate with different tails of different cytokine receptor. Depending on the cytokine receptor proteins brought together, different stat proteins bind to them. Janice kinases phosphorylate stat proteins and the STAT proteins bind together (homo or hetero). Then transit to the nucleus, firing off transcription, leading to the effector function of whatever that T cell was that secreted the cytokines. Cytokine receptors have a subunit specific for the cytokine and another that may bind to multiple. Receptor subunits come together when cytokine is present. Bring certain proteins Lead to binding site for STAT proteins that recognize specific phosphorylated jak proteins, they get phosphorylated, get fired off and are dimerized Lots of ways to form transcription factors as homo/hetero dimers Fire off transcription of lots of things

TFH IL-21 drives class switching

Make IL21 that drives class switching in B cells B cell has IL21 receptor on its surface that is being produced by TFH IL12 binds, activates JAKS and stats a bunch of transcription factors are now made. In the complex of all these proteins being made, some lead to formation of plasma cells, memory cells, AID leads to class switching. IGm or IGG3 or if IL4 is also present and binding to a site for IL4 cytokine receptor, it can class switch to Igg2a if interferon gamma is also present. If IL10 is present it can class switch to IGA. IDK. It's in the picture. Various cytokine receptors on the surface of the cells and presence of cytokines that can activate them lead to transcription of various proteins that derive some functional characteristic of that recipients cell. All carried out by cytokines that are being secreted by TH (CD4+) cells. Cytokines work through specific receptors that lead to a specific transcriptional event. Combination determines what happens to recipient cell.

Dendritic cells have to get into the lymph node

Movement of the DC to the draining LN requires activation of the DC, upregulation of CCR7 on its surface, and its homing to higher levels of CCL19 and CCL21 made in LNs DCs can enter LN through afferent lymphatic or from vasculature through HEV Best way to get cells activated- bring antigen from the infection to the lymph node. Need dendritic cells. ex: adjuvant from vaccine activates the dendritic cells, which causes minor pain or mild disease state Dendritic cells are the best. Recycle membrane a lot. Micro/macropinocytosis are beneficial for picking up debris. Activation results in pulling in processes (rounding up) and pushed into open ended lymphatic due to the increased fluid pressure. Bring antigen to lymph node. Drawn into lymph node using CCL19 and CCL21. Drawn in because dendritic cells in the process start to express receptor for chemokines (CCR7). In the lymph node, naïve T cells are coming in, and they bouncing against the dendritic cells, make contact, and sample to see if they Coming out of hev- chemokines, similar to neutrophils. Can come in as immature blood dendritic cells from bone marrow and migrate through HEV (not primary mechanism because of low amounts of CCR7) Same mechanism as T cells through the HEV basically.

Activation of CD4+ T Cells p2

NFkB- a lot is inducing proliferation in cells Ca2+ release and influx from extracellular place in innercellular calcium storage Calcium -> calcineurin -> calcineurin falls off of NFAT (transcription factor for activating T cells) -> leads to production of CD25 (cell surface protein that makes IL2 receptor high affinity, alpha chain)

Apoptosis/Programmed Cell Death

Necrosis •Cell death due to physical or chemical injury •Lyses or disintegrates leaving cell debris to be cleaned up by phagocytes •Enhanced inflammation Apoptosis (this is how cytotoxic T cells kill) •Programmed cell death in which the cell is induced to degrade itself from within (a.k.a - cell suicide) •DNA is fragmented by nucleases •Loss of membrane integrity •Vesicle shedding Prevents the release and spread of infectious particles

Effector function of CD8 T-cells

Note: No B7 signal is required for effector function!!! Kill virally infected cells Once T cell is activated for the first time, it doesn't need signal anymore. Once they are cytotoxic and go to site of infection, t cells recognize MHC1 (not expressing signal 2 anymore, only need signal 1) Dump cytotoxic chemicals.

Figure of apoptosis enzymes

Perforin, granzyme. Granzymes target multiple caspases, cleaving them and releasing the active form. Caspase 3 is penultimate apoptotic cell. When it is cleaved, active cleaves other caspases and proteins that lead to DNA fragmentation, cleaves proteins important for holding the DNA together and proteins that play a role in loosening up the membrane, lamens play a role in vesicle shedding.

CD40 ligand deficiency

Problems- B cells need CD40 signalling to be able to class switch (which led to hypogammaglobulinemia) In periphery, macrophages are bad at destroying bacteria Without CD40 ligand, nothing to bind to CD40. Don't turn up expression of interferon gamma receptors. Even if T helper cells make interferon gamma, nothing to bind to it. Deficiency leads to constant low grade infection with microbes, and antibiotics is not sufficient to cure it. This is why they do hematopoietic stem cell transplants, which doesn't always work. Formation of granulomas too.

TH2

Some stay and secrete IL4 and IL5 helping B cell development, some leave lymph node and go to mucosal tissues where they fight parasitic infections Other functions later in the powerpoint

Vaccine and Linked Recognition

Sometimes it is hard to get T cells activated to a specific peptide because the B cells won't phagocytose what it needs to to make the peptide. Vaccine is designed with a piece of the protein or peptide that you want the T cells to recognize, while the B cell recognizes carbohydrate reside on protein, allowing B cell to take in vaccine and present the peptide to activate their T cells.

The T cell/DC interaction

T cells interact with many DCs in the LN cortex, but the interaction with activated DCs via DC-SIGN stops them and strengthens their interaction Now....does the T cell recognize MHC-peptide? Molecules used to tether D cell to dendritic cells (some are LFA1, ICAM1, ICAM2, CD2 on T cells) DC-SIGN sign of activation of dendritic cells. this and its ability to bind stops the T cell for several hours so the T cell has an opportunity to recognize peptide and dendritic cell. Addition of CD2 and ability to bind stops the t cell While sampling, it uses LFA1 and CD2 (often used as a T cell marker, adhesion molecule). If dendritic cells are activated (via NK cells or binding of pathogens to TLR) they express DC-SIGN only when activated. DC-Sign adds additional cell adhesion molecule that can bind to ICAM on t cells to stop the T cells, allowing its receptors to move around on the surface and sample. Tethering allows it to test its receptor across lots of combinations. If it doesn't recognize, it pulls off and is drawn by chemotactic signal If it does, it clamps down on the cell for up to a week. The t cell receptors and mhc are activated and proliferating (lots of signals going on) but you need a second signal.

TH1 IL-12 drives TH1 skewing

TH1 cells are driven to become TH1 cells by IL12 and interferon gamma. Then express the transcription factor T-Bet Make IL12 and interferon gamma. (IFG) Primary function is activation of macrophages IL12 binds to IL12 receptor, phosphorylating JAK and family kinases, leading to phosphorylation of several STAT proteins Following one pathway down, homodimer of stat4 that is created due to IL12 binding to its receptor. One of the downstream effects is to drive interferon gamma production, which is what TH1 cells do. Another pathway: activation of stat3 leads to production of transcription factor RORgc which can also drive the T cells that are developing around the dendritic cell to become TH17 and make IL17. IL12 receptor plays a role in a bunch of this complicated mess, can bind to several different cytokines, depending upon the level of the receptor and the different cytokines different STAT proteins get phosphorylated leading to different outcomes.

Changes to DCs occur as they transport antigen

Take up antigen Endocytose or bind Express the same PRRs as macrophages and B cells Have all TLR basically When they take up the pathogen, they are activated (main way). Retract projections and turn them more circular, allowing them to migrate through lymphatic easier. Enters lymph node, extending projection so there is a large area for T cells to interact with. In lymph node near high endothelium venules are, where the T cells migrate in from (T cell rich region)

Signals from T-cell receptors, co-receptors, and co-stimulatory receptors activate naive T cells

Three-dimensional segregation of supramolecular activation clusters in T cells All proteins- CD28, T cell receptor, CD4/CD8, CD3, TCRz found in the middle of a synapse Peripheral ring and central region. P-smac: LFA1, ICAM1, Talin C-SMAC: TCR, CD2, CD4, CD8, CD28, PKC-0 Outer ring: Multiple from cell adhesion Inner ring: signal transduction

All of these interactions stop the T cell and allow it to extravasate into the LN

Very similar to Neutrophils, just different chemokines and chemokine receptor CCL19 and CCL21 draws it in. (constantly diffusing out of tissue) LFA1 clamps down in HEV. Chemokine receptor is CCR7 bind inside the lymph node once it has slowed down. Slowed down due to glycosylated residues. Push its way into the tight junction. Same mechanism as naïve B cells to go into lymph node except chemokine/chemokine receptor

Changes in Cell Surface Molecules

¡Activated T cells stop expressing L-selectin (which is used to bind to regular endothelial cells) and start expressing VLA-4 which allows them to bind to inflamed/infected endothelium.. Recognizes VCAM. Don't need to know subunit names Mucosal tissue uses different endothelium cell marker and there are different subunits When activated T cell leaves lymph node going back through vasculature, coming to the site where the infection is, it goes out to the site of infection because vascular endothelial cells (due to cytokines and inflammation) express VCAM1 (cell adhesion molecule) Activated T cells with VLA4 bind to VCAM, stopping effector T cells at the site of infection, migrating into the tissue. Different subunits and binding partner than ICAM.

Differentiation of T cells

¡Activation leads to differentiation dependent on the cytokines present ¡Daughter cells are in the same cytokine milieu around dendritic cell and thus differentiate into more of the same effector type Positive cascade driving daughter cells to take on the same phenotype Early on in immune response, major driving force for effector T cells

T-cell homing to the LN

¡As T cell enters the LN, L-selectin on its surface interacts with the vascular addressins (GlyCAM-1 and CD34) ¡Additional interactions of LFA-1 on the T cell with ICAMs on the vascular endothelial cell stops the T-cell at the HEV -Use cell adhesion molecules Glycosylated addressins, naive T cells use lectins to bind, slowing migration.

CD4+ TH2 Cells Activate B Cells That Recognize the Same Antigen

¡Cognate interactions - cell-cell interactions between B and T lymphocytes specific for the same antigen Ex: TH2 receptor, CD4 and CD40 ligand forming complex with B cell Depending on cytokine released by TH2 cell, it generally drives B cells to become plasma cells. Plasma cells secrete antibodies based on what class switching takes place, which is also dependent on cytokines. Cognate pair- antigen presenting cell and recognition cell. Cognate interaction.

Recap 1:

¡Dendritic cells carry antigens from sites of infection to secondary lymphoid tissues -Express markers for activation of T cells (DC-SIGN and B7) ¡Dendritic cells are adept and versatile at processing pathogen antigens ¡Näive T cells first encounter antigen presented by dendritic cells in secondary lymphoid tissues ¡Homing of näive T cells to secondary lymphoid tissues is determined by chemokines and cell adhesion molecules ¡Activation of näive T cells requires signals from the antigen receptor and a co-stimulatory receptor -Activated due to signal 1 and signal 2

Two ways to activate CD8 cells

¡Due to their cytotoxicity, naïve CD8 T cells require more CD28 (B7) signal to become activated. Dangerousssss. ¡Only DCs express enough B7 to activate CD8 T cells ¡Combination of two signals increases IL-2R and IL-2 expression on the T cell and it then can proliferate and differentiate -Same signalling cascade. Interacts with LCK, etc. CD8 t cells also need B7, but need way more than CD4 T cells. Killer T cells can destroy cells in your body (this is a peripheral tolerance mechanism), become anergic. More CD4 cells that are binding to the B7, highly regulating cytotoxic B cells. If the dendritic cell is infected with the virus (in the tissue), then the infected dendritic cell highly presents viral peptide (not much self peptide) and antigen specific T cells will be activated over cytotoxic T cells. Another regulatory process- CD4 and CD8 ration of like 2:1 or 3:1 Since they are using up the B7, you may not get all the cd8 activated

Humoral and Cell-Mediated Immunity

¡Humoral immunity •Antibody-mediated •Protects against bacterial toxins, bacteria, and viruses by producing antibodies against these agents •Involves TH2, TFH response, although TH1 cells can also be involved (because they are drawn out of lymph node to the site of the infection and there aren't any B cells there usually) ¡Cell-mediated immunity •Carried out by T cells •Involves TH1, TH17 response -Help macrophages and neutrophils out in the cell This took several days, innate response occurred but it wasn't enough. Dendritic cells migrate, activate T cells, then they go back out.

Important notes concerning CD8 T-cell activation

¡More stringent signal requirements to get CD8 T cells to become effector cells ¡CD4 T cells outnumber CD8 2:1 and therefore predominate the interactions with APCs in the secondary lymphoid tissues. They use up most of the B7 ¡Only when the CD8 T cell gets sufficient B7 so it can make its own IL-2 OR it gets IL-2 from adjacent CD4 cells does it become activated!

Several mechanisms activate DCs

¡NK cells can also activate DCs in the tissue, leading to changes in gene expression allowing the DCs to migrate to draining lymph nodes -NK cells in the periphery produce the IFg that activates dendritic cells, allowing them to pull in processes so they can migrate NK>Dendritic =apoptosis NK<Dendritic= activation 3rd column Early in an infection when neutrophils monocytes, and NK cells migrate to the tissue, they outnumber dendritic cells and stop the dendritic cells from functioning, stimulating innate response. If they need the adaptive immune response, dendritic cells are made in the tissue and begin to outnumber other cells, becoming active.

T cells must get to the DCs!

¡Naive T cells first encounter antigen presented by dendritic cells in secondary lymphoid tissues ¡Homing of naive T cells to secondary lymphoid tissues is also determined by chemokines and cell-adhesion molecules Signal draws them from cortex to medulla (chemotactic). Making contact with dendritic cells, looking for MHC and peptide. Stops and get activated if it does recognize. Leave through efferent lymphatic if they don't recognize anything. Driven by gradient of phospholipid expression (phengosome 1). Immature cells express receptor for S1P, moving. WOrks like a chemokine. Higher -> higher S1P. Only stop if they find MHC peptide.

T cell migration in LN

¡Naïve T cells migrate from low S1p levels around HEV to High levels in medulla ¡Activated T cells express CD69 which sequesters S1P receptors from the surface of the T cell -Stop if they come in contact with dendritic cell and get signal 1 and signal 2 because of CD60 When CD69 is turned on, it binds to receptors that causes them to migrate (recycling and removing it). Stops T cell migration in the cortical region after activation.n Causes them to stay there and proliferate more.

T Cell Activation

¡Once T cells are activated, they detach from APC: §CD8+ and TH1 T cells (and TH17) leave lymph node and go to infected sites §TH2 T cells stay in the lymphoid tissue to help activate B cells or move to mucosa where they will also help B cells §TFH cells migrate to the follicles -upon activation, express CXCR5 and home to CXCL13 made in the follicle (follicular dendritic cells), migrate towards follicle §TH17 cells migrate to sites of infection - enhance neutrophil response (expression of CXCL8 by vasculature)

Two ways to activate CD8 cells part 2

¡Other APCs have suboptimal B7 -CD4 outnumber CD8, removing the B7 signal for CD8 ¡CD4 help makes IL-2. Can bind to IL2 receptor on CD8. ¡CD8 T-cell with suboptimal CD28 signal makes high affinity IL-2 receptor. ¡IL-2 from CD4 cell activates proliferation of CD8 cell. IL2 production is really driven by CD28. Can have the high affinity for IL2 but cannot drive proliferation of more CD8, but neighboring CD4 cells can. Can use IL2 secreted by CD4, allowing it to bypass a need for a strong signal 2. Defect in central tolerance- lots of autoreactive T cells, most T cells getting signal 1 and 2 are autoreactive, increasing chance they get activated Remember: cross presentation Expresses virus (for example) on MHC1 and MHC2. Immunoproteasome formation that drives cleavage of viral proteins and production of more peptides that bind to MHC2 come from immunoproteasome from viral proteins being degraded. Preferentially put viral peptides rather than self.

What's next 1:

¡Proliferation and differentiation of activated näive T cells are driven by the cytokine Interleukin-2 ¡Antigen recognition in the absence of co-stimulation leads to a state of T-cell anergy ¡The cytokine environment determines which differentiation pathway a näive T cell takes ¡Näive CD8 T cells require stronger activation than naive CD4 T cells ¡Effector T-cell functions are mediated by cytokines and cytotoxins

Know the following TCR signaling items: (talked about in the past slides)

¡TCR:CD3 and zeta chain phosphorylated on ITAMs by Fyn ¡ZAP-70 attracted to pITAMs on zeta chain, and then is phosphorylated by Lck (which is associated with CD4 or CD8) ¡ZAP-70 activation leads to three pathways: §Ca2+ release and influx via IP3 which activates NFAT transcription factor. This ultimately produces IL-2 and CD25, which is the IL-2Ra protein. This leads to proliferation of the T cell. §Diacylglycerol leads to Ras/MAPK pathway and AP-1 transcription factor activation. This ultimately produces CD69, which sequesters the receptor for S1p and keeps T cells from leaving the LN §DAG also activates NF-kB transcription factor which leads to production of BCL-xL, an anti-apoptotic gene. ¡CD28 signaling amplifies the production of NF-kB and enhances proliferation.

Activation of T cells changes the expression pattern of adhesion molecules

¡allows effector cells to migrate to sites of infection ¡a number of the adhesion molecule changes allows the effector T cell to respond to lower levels of MHC+peptide When a cell goes from resting naive T cell to an activated T cell, changes expression (or level) of cell surface proteins Transcriptional events Can use levels to determine if it is TH1, TH2, regulatory, etc. Turn up expression of VLA4 (integrin) VLA4- family member of LFA1- bind to Icam. VLA4 allows activated T cells (after leaving lymph node to go to site of infection) to recognize a specific type of cell adhesion molecule in the vascular endothelial cell at the infection You can tell if it is TH1, TH2, etc.

Macrophage activation

¡main function of TH1 cells is to travel to site of infection and activate Macrophages ¡Mac activation requires 2 signals §IFN-g induces Macrophages to upregulate CD40 and MHC Class II §CD40L on T cells binds CD40 on Macrophages ¡Increase lysosome components ¡Process takes hours....T cell must maintain contact with macrophage -TH1 secretes lots of interferon gamma to activate macrophage, but needs help from CD40 ligand and CD40 on macrophage. -complex is T cell receptor binding MHC and a foreign peptide. In picture, CD4 helper cell binding to MHC2. Not enough signal, needs CD40 and one of those cytokines that these T helper cells make, interferon gamma. IFG binds to the receptor and the combination of all of theses signals leads macrophage to increase production of vesicles, to load those vesicles with enzymes , to build into those vesicles proton pumps so that the pH can be dropped inside those vesicles, and more factors that turn vesicles into lysosomes. Creates more lysosomes and more lysosomal enzymes, and at the same time stimulates production of more MHC2. Macrophages are better at phagocytosing the pathogen on the outside and presenting it on MHC2 to get more T cell help. End result: phagocytose and kill pathogen much better. Stimulates production of more interferon gamma receptors so they become more responsive to the interferon gamma.

Induction of Apoptosis

•CD8 T cells secrete proteins that initiate apoptosis, loaded in the lytic granules: •Perforin - protein that forms transmembrane pores in the target cell membrane •Granulysin - detergent-like proteins (polar) that embed in membrane, and aids in formation of pores (perforin) and vesicles •Granzymes - serine proteases that cause apoptosis •Cleavage of Caspase-3

Activation of CD4+ T Cells

Associated with CD4 and CD8 is a SARC family kinase which is important T cell receptors and MHCs + peptides aggregating together. FYN phosphorylates the CD4s, the ITAM units (zeta chain) on CD3 and TCRz? ZAP70 binds to the phosphorylated proteins (zeta associated protein). Recognizes phosphorylated ITAMs. ZAP70 (adaptor protein) gets phosphorylated by LCK (which can also phosphorylate the zeta chain but FYN does this, CD4 brings this in) Initiates a bunch of cascades. Need to know early part One of the cascades leads to phosphorylation of phospholipase C gamma (PLCg), which cleaves phosphytalenol bisphosphate into IP3 and DAG Releases ca2+ Within this pathway, there is activation of a mapK pathway that leads to production of transcription factor AP1 AP1 is important for downstream effect for CD+ T cells as they are getting activated. Causing increase of CD69, a transmembrane protein that is one of the earliest markers of T cell activation. 3 important transcription factors: NFKB, AP1, and NFAT. Essential for T cell to differentiate and proliferate.