Immunology

NK mechanisms of toxicity

(1) Perforin/granzymes (2) FasL - Fas (3) Cytokines (TNF) NK cells do not kill targets that express MHC class I

The TCR signaling strength (composition) = fate decision

-# number of complexes -range of the peptides available

Stages in phagocytosis

1 movement of phagocyte to microbe chemotactic signals (MDP, C) 2 attachment to the phagocyte surface mannose, C and Fc receptors 3 endocytosis of microbe into a phagosome invagination of surface membrane 4 fusion of phagosome with a lysosome microtubules involved 5 killing of microbe oxygen dependent/independent Macrophages kill organisms using reactive oxygen intermediates (ROI), nitric oxide (NO), and lysosomal proteases. These activities are localized within the phagosome/lysosome to protect the macrophage from damaging itself. These activities are upregulated by IFN-gamma. Patients with chronic granulomatous disease have a defect in phagocyte oxidase. This enzyme converts oxygen into superoxide anion and free radicals, and these patients suffer from recurrent intracellular bacterial and fungal infections.

From 1999 to 2010, the incidence of fatal drug-induced anaphylaxis in the United States increased by nearly what percent?

100%

Complement

20 distinct, but interdependent proteins, which on sequential activation may mediate protection against microbes and contribute to the inflammatory response. Complement can be activated directly by microbes (alternative pathway), or by antibodies bound to a microbe or other antigens (classical pathway). Components of the complement system can: a) Initiate (acute) inflammation, b) Attract neutrophils (chemotaxis), c) Enhance attachment of microbes to phagocytes (opsonization), and d) Kill the microbe.

CD154

AKA CD40 ligand; CD40L Activated CD4+ T cells Binds to CD40. Activates B cells, DCs, and macrophages

CD178

AKA Fas-ligand Induced on T cells and NK cells Binds to Fas and induces apoptosis on Fas+ cells; important for regulation of the immune response

CD80(B7-1)/CD86(B7-2)

AKA: b7, b7-1 B cells, macrophages, DCs Binds to CD28 and CTLA-4. Costimulator for T cell activation.

APCs

APCs (Macrophages, B cells and DCs) are able to trigger the activation of T cells that recognize the specific MHC-peptide complex via their TCR.

Activation of T cell effector function.

Activation of Naïve T cells by APC T cell activation by antigen requires 'other' plasma membrane events for activation of T cells (i.e., costimulatory molecules). These molecules are expressed by APC such as dendritic cells, which are "the cell of choice" for activating naïve T cells. Infections (and resulting cytokines) induce expression of the plasma membrane molecules, which are required for activation of T cells. The activation of T cells results in the provision of cytokines to other cells of the immune system for changes in cell biology, survival, and differentiation. Effector cells in cell-mediated immunity: Once a naïve T cell receives both the specific signal via TCR and additional co-stimulatory signals via costimulatory molecules (such as CD28), T cell activation is optimized. As a result of the activation, the T cell produces autocrine growth factors, (like IL-2, which induces clonal T cell expansion), alters cell surface molecules (so as to control trafficking in and out of secondary lymphoid organs, tissue, and blood vessels), and expresses membrane proteins that trigger other cells in the immune system (e.g., CD154). Furthermore, T cells produce cytokines that regulate the activities of many other cells of the immune system. T cells with different functions recognize pathogens in two distinct intracellular compartments. Both bacteria and viruses can replicate in the cytosol, while some pathogenic bacteria and eukaryotic parasites can replicate in the endosomes and lysosomes. Furthermore, other pathogens replicate extracellularly. The immune system has developed strategies to eradicate both intracellular and extracellular pathogens via cell-mediated immune responses. These cell-mediated immune responses many times work in concert with humoral (antibody)-mediated immune responses. The effector T cells that comprise the cell-mediated immune response can be grouped into three major categories: CD8+ cytotoxic T cells, CD4+ helper (T-helper) cells, and CD4+ T regulatory (Treg) cells. There are further subsets of Th cells that produce specific types of cytokines.

Immunological synapse

Adhesion molecules and the immunological synapse Binding of the TCR to peptide:MHC, and subsequent release of small chemotactic molecules called chemokines by the APC, results in the directional recruitment and activation of T cell surface molecules called integrins. Integrins bind to ligands on APCs, thereby forming a tight interaction between the T cell and the APC. This space between an interacting T cell and APC is known as the immunological synapse. Additional MHC molecules and TCRs are recruited into the synapse where they can interact for a prolonged period of time, optimizing signaling between the cells.

CD40

B cells, macrophages, DCs Binds to CD154; involved in activation of B cells, DCs, and macrophages

Acute phase proteins

B. Acute phase proteins are a heterogeneous group of plasma proteins (see below) important in innate defense against microbes (mostly bacteria) and in limiting tissue damage caused by infection, trauma, malignancy and other diseases. They are mainly produced in the liver, usually as the result of a microbial stimulus or in response to cytokines (IL-1, IL-6, and TNFα). Acute phase proteins maximize activation of complement and opsonization of invading microbes, and limit tissue damage. C-reactive protein (CRP) binds bacterial phosphoryl choline, activates C Serum amyloid A (SAA) activates C, acts as an opsonin Mannose binding protein (MBP) binds mannose on bacteria (opsonization), activates C Complement components chemotaxis, opsonization, enhance microbial killing Metal binding proteins removes metal ions required for bacterial growth α1 anti-trypsin, anti-chymotrypsin protease inhibitors

Monitoring the outside compartment is easy (e.g. Abs) How to monitor the inside of every cell??

Bring pieces of every protein to the cell surface where they can be examined by immune cells. How big do these pieces need to be to provide specificity? 20 amino acids, so 2 aa peptides = 20^2 = 400 combinations 3aa peptides = 20^3 = 8000 combinations 10 aa peptides = 20^10 combinations

Interferones

C. Interferons (IFNs), of which there are two types (Type I and Type II), are proteins that protect against viral infections. These molecules interfere with viral replication but also are signaling molecules between cells. Type I IFNs, IFNα and IFNβ, are produced by many different cells and interfere with viral replication by inhibiting protein synthesis in virally infected cells. Type II, immune interferon, IFNγ is mainly produced by leukocytes (T cells and NK cells) and regulates Th1 responses, increases phagocytic activity and antigen presentation. Type I, IFN-α/β - Origin all nucleated cells - Induced by viruses, some bacteria, cytokines - Functions anti-viral, inhibits cell proliferation Type II, IFNγ - origin NK cells, CD4 and CD8 T cells - Induced by TCR or NK receptor stimulation - Functions: anti-viral, activates macrophages and APC function

T memory cells

CD4 and CD8 Memory T cells in the tissues and secondary lymphoid organs: There are several types of memory T cells: central memory and effector memory. The latter phenotype of memory T cell rapidly matures to effector function and enters tissues in response to infection/inflammation. These effector T cells can exist as either short term effectors (they die within days) or longer term (weeks to months) effectors. Some effector memory CD8 T cells stay in the lungs after flu virus infection. Thus like phagocytes, T cells can be in tissues in advance of infection if they were called to the tissues previously, i.e., it's your second flu infection. Effector memory T cells tend to proliferate less and make cytokines for effector function. Central memory T cells tend to proliferate rapidly in second encounters with MHC/peptide and thus increase the number of effectors, which includes CD4 T cells that help B cells make antibodies

T reg

CD4+ Regulatory T cells (Tregs): Tregs are considered CD4+ T cells, but they are not considered helper T cells because they suppress immune responses. They are important because they help to maintain peripheral tolerance to self-proteins. Regulatory T cells that suppress immunity in the periphery can suppress cell-mediated immunity and humoral immunity. Regulatory T cells mediate suppression through a currently unknown contact-dependent mechanism, as well as through the elaboration of cytokines (like IL-10 and TGFβ) that reduce inflammatory responses. Their importance is underscored by mutations in FoxP3 (a transcriptional repressor) that Tregs express. Loss of FoxP3 leads to autoimmunity of a number of organs.

CD4 cells

CD4+ helper T cells: During the course of T cell activation, T cells differentiate, and the spectrum of cytokines that they produce becomes restricted. Initially, early activated, mature CD4 helper T cells are called Th0 cells, then depending on a variety of environmental factors (cytokines, affinity of MHC interaction, etc.) they can differentiate to more specialized effector cells, called Th1, Th2, or Th17 cells. These cell types are CD4+ T cells, but they produce different spectrums of cytokines. However, all CD4+ T cells recognize MHC class II peptide complexes. Th1 T cells predominantly mediate inflammatory responses (e.g., activate macrophages) by producing the cytokines IFN-g and TNF. IL-12 is the cytokine associated with development of Th1 T cells. Th2 T cells help B cells proliferate and secrete certain types of Abs by producing IL-4, IL-5, and IL-13. IL-4 is the cytokine associated with induction of Th2 T cells. More recently a subset of inflammatory helper T cells has been identified that do not behave like either Th1 or Th2 cells. Because these cells produce large amounts of IL-17, they have been termed Th17 cells. Th17 cells are believed to play a role in autoimmunity. A main function of Th17 cells is to recruit neutrophils.

Th cells

CD4+, produce many cytokines, central role in activation of the immune system, infected by HIV

CD8 cells

CD8 cytotoxic T cells (also called cytotoxic T lymphocytes or CTL): CTL play a critical role in host resistance to pathogens that live in the cytosol of cells, such as viruses. These CD8+ T cells can directly recognize, then kill, cells harboring such pathogens through recognition of MHC class I molecules that have been 'loaded' with peptides derived from these pathogens. Cytotoxic T cells kill target cells by various mechanisms. CTLs lyse the target cells they have recognized peptide:MHC class I by the release of granules that contain proteins that create pores in the target cell. CTLs can function by insertion of perforin and granzymes into the target cell membrane. With the integrity of the cell membrane destroyed, the target cell dies through apoptosis. CTLs can also kill by binding to target cells and releasing factors (like TNFα, IFNγ) or expressing cell surface molecules such as Fas-L (which binds to Fas on target cells). These molecules bind trigger apoptosis in target cells. The CTL can trigger this event in a brief period of time, then move on to the next target.

CTLs:

CD8+, produce some cytokines, kill other cells

CD8

CTLs, MHC class I reactive T cells Binds to MHC class I and helps in signaling for MHC I reactive T cells

Cells of the innate immune system activate adaptive immunity.

Cells infected with viruses will produce IFN-α or IFN-β (Type I interferon) that will trigger an antiviral state in nearby cells and activate NK cells to become enhanced killers of virus-infected cells. Thus, the innate immune system can trigger other effector responses to enhance host defense against the type of infection that occurs.

How does the immune response remove the pathogen?

Central tolerance Lack of stimulation signals Inhibitory proteins T regulatory cells Negative feedback

Regulation

Central tolerance Lack of stimulation signals Inhibitory proteins T regulatory cells Negative feedback

Innate effector molecules

Complement Proteins Acute Phase Proteins C-reactive protein Serum amyloid protein mannose binding protein metal binding proteins - The first three acute phase proteins bind to bacteria Metal stalls bacterial growth Interferons Type 1 (alpha/beta) Type 2 (gamma) Others Collectins anti-bacterial peptides (defensins)

Collectins and defensins

D. Other molecules. Collectins are carbohydrate-binding proteins that act as opsonins, i.e. they bind to a microbe and make it easier for a phagocyte to ingest and kill the microbe. Receptors for 3 collectins on macrophages facilitate the removal and destruction of the collectin bound microbe. Peptide antibiotics (also called defensins) are produced by a wide variety of cells including epithelial and phagocytic cells.

Eosinophils

Eosinophils are granular leukocytes that attack parasites too large to be phagocytosed, and kill them by releasing a toxin, major basic protein. Eosinophils contain granules that contain powerful mediators of inflammation They are part of the innate immune system Eosinophils have been associated with allergic diseases and defense against extracellular parasites Eosinophil production is induced by IL-5 in the bone marrow

CD3

Expressed in all T cells Associates with the TCR and mediates TCR signaling

Secretions at epithelial surfaces

Eyes - Lacrimal glands (tears) Lysozyme, IgA and IgG Ears - Sebaceous glands Wax (cerumen) Mouth -Salivary glands Digestive enzymes, lysozyme, IgA, IgG, lactoferrin Skin - Sweat/sebaceous glands: Lysozyme, high NaCl, short chain FAs Stomach -Gastric Juices Digestive enzymes (pepsin, rennin), acid (low pH - 1-2)

DCs trafficking

How does the adaptive immune arm know that a pathogen is present?lymphatics are a one way street, dcs can enter through afferent lumphatic vessel... Since each individual has billions of lymphocytes each with different antigen specificity, how is the specific lymphocyte supposed to meet up with its antigen on an APC near the site of infection? The answer is the system of draining lymph nodes throughout the body. Lymphocytes constantly recirculate around through the different lymph nodes. Dendritic cells (DCs) pick up antigen in the periphery and bring it to the local lymph node via lymphatic drainage. Primary (or central) lymphoid tissue is where lymphocytes develop and become competent to respond to antigens (e.g. thymus and bone marrow). Secondary (or peripheral) lymphoid tissue is where mature lymphocytes reside and where adaptive immune responses are initiated.

Cells of the innate immune system work together

INNATE IMMUNE CELL NETWORK Innate immune cells can activate responses in autocrine and paracrine manners. Macrophages can produce cytokines such as TNF-alpha that will activate its own production of IL-12 and help in the recruitment of PMNs. IL-12 will activate NK cells to produce IFN-gamma that will further activate macrophages to become more effective killers of bacteria. IL-12 also induces the upregulation of IL-2 receptors on NK cells and allows them to proliferate to IL-2. Thus, the different cells of the innate immune response can activate each other to attack microorganisms prior to the generation of an adaptive immune response. It is important to remember that TNF-alpha, IFN-gamma, and IL-12 are also able to activate T cell immunity.

What antibodies (GAMED) activate complement?

IgG and IgM

Tolerance

Immunological unresponsiveness to self-antigens Lymphoid cells with receptors for self-antigens are not allowed to develop into mature cells Those cells that recognize self-antigens that "sneak through" are not allowed to be activated

Receptor crosslinking

In order to stimulate cells through their antigen-specific receptors, it is necessary to trigger several of the receptor molecules on the cell surface at the same time. The ligands for the immune receptors are typically displayed on the surface of antigen presenting cells, and this allows many of the receptors to bind to their cognate ligands at the same time. This results in the receptors localizing near to each other on the cell surface, which brings kinases and other signaling molecules in close proximity within the cell. The process of receptor cross-linking leads to intracellular signaling that begins lymphocyte activation. Antibodies can also be used to cross-link receptors and mimic this process

Innate lymphoid cells

Innate Lymphoid Cells. There are a number of lymphoid cells that are T cells, T cell-like, NK-like, or B cell subsets that express unique receptors with innate-like recognition, that is molecules common to microbes. These cells are often found in mucosa, peritoneum, liver and other tissues. They provide a part of the rapid defenses against infection.

Immune receptors

Innate: Pattern recognition receptors: innate receptors for bacterial/viral components (many cell types) Receptors for self proteins: NK receptors, cytokine receptors Fc receptors: bind to antibody (Ig) (NK, Macrophage, PMNs, Eos, Mast cells) Adaptive: Immunoglobulin (Ig): specific for one antigen (B cells). When secreted it is referred to as an antibody. T cell receptors: specific for one peptide + MHC (T cells)

TNF-alpha

Lymphocytes, macrophages, mast cells Targets: Many cells Effects: Proinflammatory; endothelial activation, shock, acute phase proteins, PMN activation

MHCI pathway

MHC I Pathway Peptides from intracellular antigens are presented to CD8 T cells on MHC I molecules. MHC I molecules are found on all nucleated cells and constantly present cytoplasmic antigens to T cells. These antigens include proteins from intracellular pathogens as well as normal self proteins. However, only professional antigen presenting cells that have encountered molecular stimuli from pathogens in the periphery are capable of providing costimulation, together with MHC:peptide, for effective T cell priming. To access the MHC I pathway, protein antigens in the cytoplasm must first be targeted to the proteasome for degradation. The proteasome is a multi-subunit protein complex that cleaves ubiquitinated proteins into peptides of 8-10 amino acids. Professional APCs interchange proteasome subunits to form an immunoproteasome, which is more efficient at generating peptides that can bind to MHC I. Short peptides are then transported into the ER by a molecule called transporter associated with processing (TAP). Peptides are loaded onto newly-synthesized MHC I molecules in the ER, and are transported to the cell surface through the Golgi apparatus and exocytic vesicles.

MHC

MHC I are found on all nucleated cells and present peptides from internal proteins. MHC II are found on APCs and present peptides from externally-derived proteins.

MHC structure and peptide binding

MHC I is comprised of a single polymorphic chain in association with a small invariant chain called beta-2 microglobulin. The peptide-binding cleft is closed on either end and can only accommodate peptides ~8-10 amino acids in size. MHC II is comprised of an alpha chain and a beta chain, which are each encoded by different genes in the MHC locus. The peptide-binding cleft is open on either end and can therefore accommodate peptides with a wide range of sizes (~8-30 amino acids). MHC molecules have polymorphisms on the floor of their peptide binding clefts, which enables peptides to be bound only if they have anchor residues with specific characteristics (i.e. charged, hydrophobic, etc) that are compatible with that MHC molecule. MHC molecules will each bind many different peptides, however if the MHC molecules from a cell are unable to bind to any peptide from a particular antigen, then the host will be unable to mount a T cell response to that antigen. If a peptide binds a particular MHC it is said to be "restricted" to that MHC molecule.

MHCII pathway

MHC-II is predominantly expressed by APC's therefore CD4 T cells predominantly respond to APC's MHC II Pathway Peptides from extracellular antigens are presented to CD4 T cells on MHC II molecules. MHC II molecules are, for the most part, only found on professional antigen presenting cells, although there are some exceptions to this rule. Antigens access the MHC II pathway by first being phagocytosed/endocytosed into acidified endosomes. Immature macrophages and dendritic cells (DCs) are highly phagocytic while in peripheral tissues. Phagocytosis is an indiscriminate process, leading to uptake of extracellular pathogens, proteins, as well as dying normal cells and their fragments. Therefore it is again important to note that only APCs that have encountered molecular signals from pathogens in the periphery upregulate costimulatory molecules necessary for T cell priming. On the other hand, B cells acquire antigen in an antigen-specific manner by only phagocytosing antigen that binds to the B cell receptor (BCR). Due to the high-affinity interaction of the BCR with its cognate antigen, B cells are particularly adept at presenting antigen at low concentrations. This also means that a B cell only presents peptides to CD4 T cells that recognize linear peptide fragments of the same antigen that the B cell recognizes in its native conformation. Following uptake of pathogens/antigens into acidified endosomes, proteases break antigens down into smaller peptide fragments comprising a wide range of sizes. These endosomes/lysosomes then fuse with vesicles containing MHC II molecules. It is in these specialized vesicles that peptide fragments are loaded onto MHC II molecules. Because MHC II molecules are very unstable in the absence of peptide fragments, they are transported in association with a molecule called the invariant chain. Subsequent cleavage of the invariant chain leaves behind a peptide fragment called CLIP, which occupies the MHC II peptide-binding groove, but is exchanged for peptide antigen within the vesicle. This process is facilitated by an MHC-like chaperone molecule called DM. The MHC II peptide complex is then transported within the vesicle to the cell surface for presentation to CD4 T cells.

IL-1

Macrophages, B cells, NK cells (Endothelium, fibroblasts, astrocytes, etc...) Targets: T cells, B cells, macrophages, endothelium Effects: lymphocyte activation, macrophage stimulation, incr. leukocyte - endothelium adhesion; acute phase proteins, fever

Activation via these innate receptors leads to upregulation of co-stimulation molecules that provide "signal 2" to lymphocytes. These receptors are often referred to as pattern recognition receptors (PRR).

Mannose receptors are expressed on macrophages, dendritic cells and endothelial cells. They bind to mannosyl/fucosyl carbohydrates on microbes, phagocytose the microbe and then process and present microbe peptides on MHC molecules, thus inducing specific anti-microbial T and B cell responses. Other receptors. CD14 is a molecule expressed on macrophages that binds LPS on gramnegative bacteria, and facilitates destruction of the microbe and induction of secretion of cytokines involved in triggering adaptive immune responses. In addition, scavenger receptors on macrophages recognize carbohydrates and lipids in bacterial and yeast cell walls. Toll-like receptors (TLRs) are located at the cell surface or in cellular vesicles and recognize molecular patterns from different groups of pathogens. They signal the presence of a pathogen and trigger the expression of co-stimulatory molecules and cytokines important in the development of adaptive immune responses. NOD proteins have a similar function, but they are located in the cytoplasm. mannose receptors mannosyl/fucosyl structures; macrophages, endothelial & dendritic cells CD14 LPS macrophages scavenger receptors carbohydrates or lipids macrophages toll-like receptors LPS, peptidoglycan, glucans, APCs, macrophages, lymphocytes, other cells

Macrophages/Location

Monocytes Blood stream Kupffer cells Liver Mesangial cells Kidney Alveolar macrophages Lungs Microglial cells Brain

IL-12

Monocytes, APCs Targets: Th1 cells, NK cells Effects: Differentiation of Th1 cells, activation of NK cells

IFN-alpha/beta

Most cells Targets: most cells Effects: Antiviral; increased MHC class I expression

IFN-gamma

NK cells, T cells Targets: Macrophages, B cells, endothelium, DCs Effects: Increased MHC class II expression, macrophage activation, IgG class switching, suppress Th2 cells

Negative selection

Negative Selection: After thymocytes pass the positive selection phase, they are interrogated to determine if their TCR binds too strongly to MHC class I/peptide or MHC class II/peptide complexes. T cells with strong binding TCR might be auto-reactive (they may be activated by selfantigen in the periphery), and thus cause autoimmunity. Thymocytes expressing a TCR that binds strongly (high affinity) to MHC peptide complexes are induced to die via apoptosis. Negative selection happens in the medulla or the medullary/cortical junction. AIRE (autoimmune regulator) is a transcriptional regulator that allows expression of 'non' thymic genes that provide peptides for MHC class I molecules and subsequent negative selection. Without AIRE and proper negative selection, children present with multiple autoimmune diseases. Negative selection is not perfect and a percentage of self-reactive T cells do enter the periphery, where they may cause autoimmune disease. The good news is that most of these cells are silenced through a process called peripheral tolerance, thereby preventing autoimmunity.

Inhibition of T cell activation by CTLA-4

Once T cells become activated they begin to express the inhibitory molecule CTLA-4. Like CD28, CTLA-4 also interacts with B7 on APCs. However, CTLA-4 is of higher-affinity than CD28, therefore it is able to out-compete CD28 for binding to B7. Upon engagement of CTLA-4 with B7, T cells receive an inhibitory signal, which can dampen or terminate T cell responses. This is one method for controlling T cell activation after an infection has been cleared.

Cross-presentation of peptides by MHC I

Performed only by Dendritic cells Significance: CD8 T cells can respond to both extracellular and intracellular pathogens Significance All nucleated cells present Ag on MHC-I, which enables them to be recognized by CD8 T cells Only professional APC's have costimulatory "Signal 2" which enables them to prime CD8 T cells So how can you prime a CD8 T cell response against an intracellular pathogen that does not infect APC's? Probably more important in cancer than viruses. As an exception to the above rules, it has more recently been discovered that dendritic cells are capable ofshuttling antigens from the extracellular compartment into the MHC I pathway. It is thought that this occurs with the help of molecules or leaky vesicles that transport proteins and peptides from the lysosome into the cytoplasm. Antigens can subsequently be targeted to the proteasome for presentation on MHC I. Cross presentation is crucial for priming a CD8 T cell response against pathogens that don't directly infect APCs. For example the flu virus only infects lung epithelial cells, but these cells do not have the costimulatory molecules necessary to prime CTL responses. DCs can phagocytose infected cells and present flu antigens to CD8 T cells, thereby priming CTLs that can recognize and kill infected lung epithelial cells. Protein just gets dumped into cytosol and then goes through the MHC I pathway. Relevant in cancer - cancer cell has MHCI and can be recognized by Tcell but cannot do costimulation. How can we activate them? DCs get the peptides from the cancer and presents them to T cells, which can then attack the cancer cells.

Platelets

Platelets, when activated, release mediators that initiate complement activation leading to attraction of leukocytes. Platelet granules contain chemokines and growth factors, and platelets can be an active part of the innate immune response. Erythrocytes bind small immune complexes and help remove them from the circulation.

Are mast cells monoclonal or polyclonal for IgE?

Polyclonal. Normal immune system is seeing all different parts of same antigenic molecule. So every immune response is polyclonal with multiple types of B cells being activated.

Positive selection

Positive Selection: Thymocytes that have rearranged a TCR that can bind weakly to its 'selecting element' (MHC class I or MHC class II plus self peptide) induce TCR-mediated signals (low or different signaling components, e.g., Ras MEK, ERK) that spare the cell from death. Said another way, TCR ligation with a certain biochemical gestalt allows thymocytes to survive: this is termed positive selection. Thus, the T cell repertoire is first selected for cells that can weakly bind to MHC + self peptide that the host expresses. Thymic epithelial cells in the cortex are responsible for positive selection. If T cells fail to find an appropriate peptide:MHC in the thymus, they will die from neglect (i.e., the T cell needs signaling from the TCR in order to express genes needed for survival).

Costimulation (Signal 2)

Professional APCs also provide a second signal to T cells, known as costimulation or "Signal 2". Costimulation is provided by interaction between B7 on APCs with CD28 on T cells. B7 is only expressed by professional APCs that have recently been activated or matured by encounter with pathogens or other inflammatory stimuli in the periphery. When T cells encounter cognate peptide:MHC on a cell that is not a professional APC, they receive Signal 1 without Signal 2. This leads to T cell inactivation, which is also called anergy. Induction of anergy is one mechanism for preventing T cells that may recognize self antigens from attacking uninfected tissues.

DCs

Rare cells. Able to mediate robust activation of T cells Immature DCs sit in tissues and phagocytose material. They are poor at stimulating T cell activation. After activation (by bacterial products & cytokines), DCs stop phagocytosing material, move to local lymph nodes, and upregulate MHCI and MHCII that trigger T cells. There are various sub-types of DCs that differ in the abilities to trigger specific T cell types. Langerhans cells in skin Interdigitating cells in lymph node T cell areas Follicular DCS in B cell follicles of lymphoid tissue DCs can transfer antigen to other DCs in the lymph nodes and promote activation of antigen-specific T lymphocytes DCs are currently being explored as a means to stimulate specific immunity (immunotherapy)

Immune activation requires what two signals?

Signal one is via the immune receptor (Ig or TCR), and signal two is provided by a "costimulatory" signal. This costimulatory signal is induced by the presence of microorganisms or their products.

Superantigens

Superantigens are a class of antigens - often derived from bacteria - that cause non-specific activation of T-cells. Superantigens interact with the Variable region on the Beta chain of the T-cell Receptor, leading to antigen-non-specific T cell activation. Since these superantigens bind to all TCRs (regardless of the variable region specificity), they lead to polyclonal activation. Whereas a typical antigen:MHC may activate ~0.001% of the body's T-cells, superantigens have been shown to activate > 80% of the total Tcell compartment. Superantigen-activated T cells release large amounts of cytokines, launching an uncontrolled immunologic cascade that can impart significant morbidity or mortality. Toxic shock syndrome is an example of a bacterial superantigen-mediated disease.

CD28

T cells Costimulatory molecule for T cell activation. Binds to CD80 and CD86

IL-2

T cells Targets: T cells, NK cells Effects: T cell and NK cell activation and proliferation

How are T cells able to "see" antigens?

T cells do not recognize native antigen. For effective priming, T cells must first see peptide antigen presented by major histocompatability (MHC) molecules on antigen-presenting cells (APCs). There are three major subsets of APCs known as "professional" APCs due to their constitutive expression of both MHC I and II molecules: dendritic cells (DCs), macrophages (Mφs) and B cells. Dendritic cells are the predominant cell subset responsible for T cell priming. APC's acquire protein antigen and process it into short peptide fragments for presentation to T cells.

IL-10

T cells, Th2 cells Targets: Th1 cells Effects: Inhibition of cytokine synthesis and macrophage function

TNF-beta

T cells, monocytes (most cells) Targets: Most cells Effects: Inhibits cell activation, anti-inflammatory, promotes IgA secretion

CD4

T helper cells, MHC class II reactive T cells, monocytes, macrophages Binds to MHC class II and helps in signaling for MHC II reactive T cells. Binds to HIV

IL-6

T, B, macrophages (endothelium, fibroblasts, astrocytes, stromal cells, etc...) Targets: T, B, macrophages Induces acute phase response; regulates Th17 differentiation, and proliferation of antibody producing cells

Th1 - CD4 cells

Th1 CD4 T cells: The protective response to a variety of intracellular pathogens is dependent upon so-called inflammatory T cells, or Th1 T cells. For example, the immune response to mycobacterium is severely diminished if the host cannot produce IFN-γ and TNF-α, which are derived in large part from CD4 T cells of the Th1 phenotype. This is because, in the absence of these mediators, infected macrophages cannot become completely activated to kill the pathogens. Another important feature of the Th1 T cell is that when it becomes activated it produces cytokines that assist in the recruitment of macrophages to the site of infection. Th1 T cells make two hematopoeitic growth factors: granulocyte-macrophage stimulatory factor (GM-CSF) and IL-3. These cytokines increase the production and release of macrophages from the bone marrow into the blood. Second, TNF released by the Th1 alters the surface properties of endothelial cells to promote the adhesion of macrophages at the site of infection. The coordinated production of these mediators allows the infiltration of T cells and macrophages to the site of inflammation, where they can interact, leading to the elimination of pathogens. The IFN-g produced by Th1 cells also prevents the induction of Th2 cells and promotes the production of cytokines that increase Th1 cell differentiation. This is thought to be important as a positive feedback loop to reinforce the production of Th1 cells

Th2 - CD4 cells

Th2, CD4 T cells: The production of Abs to most antigens requires the participation (help) of T cells. The T cell most effective at inducing production of Abs from B cells is the Th2 cell. Th2 cells are able to induce (by cytokines and PM-associated receptor events) B cells to produce Abs, switch the isotype of Abs being produced, and promote affinity maturation of the Abs. (The new nomenclature uses follicular helper T cells for T cells that accomplish this in the germinal centers.) The reason for their proficiency is due to the profile of cytokines that they produce. However, in addition to the production of soluble cytokines, Th2 cells also express cell surface molecules (e.g., CD40 ligand) that directly engage the B cell's CD40 molecule, which trigger many of the events associated with B cell biology. The production of different cytokines by the T cells controls the distribution of Ig isotypes that are produced. For example, high levels of IL-4 induce B cells to produce IgE Abs. The IgE antibodies will bind to mast cells via an Fc receptor, which when triggered (IgE crosslinked by antigen) causes the mast cells to secrete a variety of mediators, including IL-4, which promotes Th2 cell induction. Thus, there is a positive feedback loop for Th2 cell differentiation too. Cytokines from Th2 cells also induce the production of eosinophils. IL5 is a growth and differentiation factor for eosinophils. Eosinophils are important for combating infection with helminths like Schistosoma mansoni (liver fluke) by the release of toxic mediators and pharmacologics that make the parasite's life difficult due to changes in the immediate environment. Eosinophils also play a major role in tissue remodeling in severe asthma.

IL-4

Th2, granulocytes Targets: Wound-healing (alternatively activated) macrophages Effects: tissue repair.

IL-5

Th2, mast cells Target: IL-5 stimulates B cell growth and increases immunoglobulin secretion. It is also a key mediator in eosinophil activation.

MHC locus and gene expression

The MHC locus, found on chromosome 6, encodes MHC I and II molecules. In humans, this is called the Human Leukocyte Antigen, or HLA. The HLA encodes three MHC I molecules and three MHC II molecules, but humans have two copies of this chromosome (one from each parent) therefore everyone has six MHC I molecules and six MHC II molecules. Twelve MHC molecules may not seem like very many, although the fact that MHC molecules are highly diverse among the human population ensures that the population as a whole will be able to mount a protective T cell response to any particular pathogen. MHC molecule expression is co-dominant, meaning that APCs express all MHC molecules simultaneously.

Thymic selection

The Process of Thymic Selection: After entry into the thymus, thymocytes initiate TCR (T cell receptor) gene rearrangements of the tcr loci, resulting in a cell surface expressed TCR that can be selected (or not). Developing thymocytes express both CD4 and CD8 molecules on their plasma membrane. These cells are called double positive (CD4+CD8+) thymocytes. However, only a few of the double positive thymocytes mature to single positive T cells (expressing only CD4 or CD8) because of the high rate of death in the thymus. The structural biology of the TCR permits selective binding of peptide-bound MHC class I or MHC class II molecules; i.e., not every TCR binds all possible MHC/peptide complexes. The folded TCR chains position the CDR regions (the hypervariable regions in the alpha and beta chains of the TCR) so the specificity of binding is possible because of the gestalt molecular signature of the MHC plus peptide. The result of TCR ligation to peptide:MHC is signal transduction in the T cell, which results in gene activation; this gene expression is essential for thymocyte viability during the maturation process. (Recall: in the periphery, T cells use CD4 and CD8 that bind MHC class II and MHC class I, respectively, as co-receptors to enhance/stabilize TCR signaling.) By convention, CD4 or CD8 expression on a cell that also has a TCR specifies a T cell that will bind either MHC class I (CD8) or MHC class II (CD4).

How do antigens get to lymph nodes?

The lymph nodes and spleen are the primary sites of T cell priming by APCs. The spleen acts as a filter for blood borne antigens, which are taken up by resident APCs for presentation to resident T cells. On the other hand, antigen in peripheral tissues needs to be taken by dendritic cells to lymph nodes for presentation to T cells. DCs in peripheral tissues are considered to be in an immature state. Here they move through tissues and phagocytose materials indiscriminately. When molecular signals from pathogens, also known as pathogen-associated molecular patterns (PAMPS) are encountered by DCs, their maturation is triggered. As a result, DCs lose molecules for adhesion in tissues and enter the lymphatics. They also lose their phagocytic capacity, upregulate MHC molecules, and begin to express high levels of costimulatory molecules. Once in the lymph nodes, these matured dendritic cells are capable of priming resident T cells.

Neutrophils or PMNs

The neutrophil (also called a polymorphonuclear cell or PMN) is the major white cell (leukocyte) in the blood, where it searches for invading microbes. PMNs possess very powerful weapons that can kill bacteria very effectively. They contain granules that contain enzymes and other molecules to kill bacteria. As PMNs die, they release histones and other components to form Neutrophil Extracellular Traps (NETs) that trap and kill microorganisms. Neutrophils move rapidly through the blood until they receive a signal to move into tissues. This signal is the expression of selectin molecules on endothelial cells. Selectins are induced on endothelium due to local inflammation, for example by TNF-alpha produced by a tissue macrophage, and selectins act as a flag to neutrophils that their help is needed at a particular location. The neutrophils bind to selectins via selectin ligand that slows their movement down so that they roll along the endothelium. As neutrophils roll, they become susceptible to signals that indicate that inflammation is ongoing nearby (e.g. C5a or LPS). If they encounter such signals, the PMNs quickly upregulate integrins on their cell surface that lock the PMNs in place by binding to ICAMs (intercellular adhesion molecules) on the endothelium. These PMNs are ready to move into the tissue by chemotaxis 6 induced by specific chemokine gradients. The PMNs move along these gradients to the site of inflammation where they can kill the invading microorganism. Neutrophils account for a large number of total leukocytes They are part of the innate immune system. They can be rapidly produced from the bone marrow. They have a short life span (hours/days) and contain granules that can kill bacteria. When neutrophils die, they can release NETs that trap and kill microorganisms. Neutrophils express FcRs and can mediate ADCC, release mediators of inflammation, and phagocytose material.

How does T cell trafficking regulate immune cell infiltration of lymphoid and non-lymphoid organs?

The reason why the mechanisms of lymphocyte trafficking and recirculation exist is to maximize encounters with antigens by survey of the different tissues for infections that generate foreign antigens that make it to secondary lymphoid organs (SLO, e.g., spleen, lymph nodes, Peyer's patches). This is the raison d'être for the lymphatic system and SLO. Similarly you need effector cells of the immune system to enter infected or inflamed areas; you need highways, signposts, and exit ramps to get to the areas that require lymphocyte or other white blood cell effector function. Recirculation of lymphocytes via blood or lymph to SLO is a means to optimize the encounter of T lymphocytes with specific antigenic peptides bound to MHC molecules. Some free antigen and dendritic cells with acquired foreign antigens travel via lymph to a lymph node from an infected tissue that the node drains. As in the thymus, T cells 'crawl' through the node, physically contacting APC (DC and later B cells) through their TCRs. If binding of MHC/peptide by TCR if accompanied by costimulation the T cell is activated. The T cells go to specific locations, and there is also an order to interactions with DC and B cells. T and B cells that have not yet encountered 'their' specific antigen are called "naive cells" and do NOT have to remain in one secondary lymphoid organ, but can circulate around the body until they recognize their specific antigen and become activated. If no recognition occurs, the life span of naïve B and T cells is limited. More T and B cells are being generated and thus the chance for other T cells to be selected keeps the diversity available in addition to what has been activated in the periphery (we call these memory cells).

tolerance

There are two main classes of tolerance: Central Tolerance: The process by which self-reactive T lymphocytes can be clonally deleted in the thymus. Encounter of antigen (strong signaling via TCR) during the development of lymphocytes can induce apoptosis (i.e., negative selection) of T cells that bind to self-antigen. Removal of self-reactive lymphocytes as they develop in primary lymphoid tissue is called central tolerance. Peripheral Tolerance (T cell anergy): Autoreactive T cells that escape deletion in the thymus and emigrate to the periphery can cause autoimmunity. Because the self-antigen they recognize may only be expressed on non-professional antigen-presenting cells (APCs), these autoreactive T cells are not typically activated to respond (i.e., no costimulation is present on non-APC). However, if selfantigen is presented on professional APCs, mature autoreactive T cells in the periphery can be triggered to respond, leading to autoimmunity. (The conditions for this will be discussed in the autoimmunity lecture.) [[recognition of self antigen; engagement of inhibitory receptors (e.g. CTLA-4)] There are several mechanisms of peripheral tolerance: 1. Ignorance: Auto-reactive T cells are not activated in tissues where they are excluded (immune privileged tissue like testes, and even epidermis where naïve T cells do not routinely circulate) 2. Anergy: Auto-reactive T cells that engage peptide:MHC in the absence of co-stimulation (for example, on a non-APC) will become non-responsive. 3. Regulatory T cells (Tregs), usually expressing CD4, CD25 (IL-2 receptor), and the transcription factor Foxp3. These cells are involved in the normal process of immune shutdown after elimination of antigen, as well as suppression of auto-reactive T cells. [[suppression of CTL and Th function]]

Describe the selection of the T cell repertoire in the thymus

Thymocytes, the precursors of mature T cells, migrate from the bone marrow via the blood to the thymus (a primary lymphoid organ) where they develop into mature T cells. A mature T cell is defined by its T cell receptor (TCR) which is used to recognize (bind) antigen and MHC. There is an order to thymocyte development that occurs in different locations within the thymus. The process of positive and negative selection selects an appropriate set of T cells (the "T cell repertoire") to recognize peptides bound to MHC class I or MHC class II. A small fraction of thymocytes leave the thymus -- most die within the thymus. The largest loss of cells is due to failure to express a usable TCR (thus, the lack of something to be "selected" by). There is also dropout due to negative selection (see below). T cells that leave the thymus are mature but naïve, meaning they are capable of being activated by APC but have not "seen" their cognate antigen (peptide/MHC) presented by an APC.

T/F: T cells monitor intracellular compartments for antigens that are derived from pathogens. This is in contrast to B cells and antibodies, which are only capable of monitoring extracellular compartments.

True

macrophages can change their function in response to environmental stimuli

True Macrophages can differentiate in response to innate and adaptive signals to have unique functions. Macrophages can be stimulated to be activated, pro-inflammatory cells that efficiently kill microbes through phagocytosis. Macrophages can be stimulated to become wound-healing cells that produce 5 extracellular matrix, alter the production of cytokines, and suppress the expansion of nearby lymphocytes. Regulatory macrophages often arise during the late stages of an adaptive immune response and limit inflammation and dampen the immune response. It is important to remember that macrophages can change their function in response to environmental stimuli.

4 types of hypersensitivity reactions

Type I (atopic or anaphylactic): immediate, IgE and mast cell activation, soluble Ag Type II (cytotoxic - ADCC): IgM, IgG (both activate complement!), Ag on cell surface Type III (immune complex): IgM, IgG, C' Ag is soluble, influx of neutrophils and release of mediators Type IV (delayed - has nothing to do with antibodies): delayed, T cells, chemokines and cytokines

Cytokine signals: The importance of IL-2

Upon effective priming by an APC, T cells begin to produce cytokines. Cytokines are a category of protein signaling molecules that, like hormones and neurotransmitters, are used extensively in cellular communication. Historically, the term "cytokine" has been used to refer to the immunomodulating agents (interleukins, inferferons, etc.). Cytokines are produced by all types of immune cells, but are also produced by other cells in the body. IL-2 is one of the earliest cytokines produced by T cells. It is predominantly produced by CD4 T cells, but can also be produced in small amounts by CD8 T cells. IL-2 acts in an autocrine fashion (on the cell that makes it) and in a paracrine fashion (on neighboring cells) to induce T cell proliferation. Proliferation of a single T cell is called clonal expansion. All T cells constitutively express the low-affinity IL-2 receptor. Upon activation, T cells begin to express the highaffinity IL-2 receptor, which enhances their ability to respond to IL-2.

NK cells

VII. NATURAL KILLER (NK) CELLS (also called large granular lymphocytes or LGLs) are found in many tissues of the body and in the blood circulation. NK cells produce and release cytokines, including IFNγ, that are important in protection against viruses and tumors. NK cells can kill cells via perforin containing granules, Fas-Fas ligand, or TNFα release. NK cells express activating receptors that bind to molecules found on stressed cells, virus infected cells, and tumor cells. When these receptors bind their ligands, they turn on NK cell killing and cytokine production. NK cells also express receptors that bind to MHC class I and shut off NK cells. These inhibitory receptors are believed to act as a fail-safe mechanism to prevent NK cell activation against normal cells. Many tumor cells and virus infected cells down-regulate their expression of MHC class I (to avoid T cell recognition) and thus become susceptible to NK cell mediated killing. Lymphocytes that do not express TCR or Immunoglobulin Also called Large granular lymphoctyes, CD3- CD56+ Important for responses to viruses (herpes family) and activation of macrophages NK cells are activated by IFNa/b, IL-12, among other cytokines Produces cytokines such as IFN-g, TNF-alpha, GM-CSF. These cytokines activate macrophages Cytotoxic granules are used to kill other cells Most NK cells express FcRs and mediate antibody dependent cellular cytotoxicity (ADCC)

Mast cells and basophils

VIII. MAST CELLS AND BASOPHILS have similar morphology and functions. Basophils are in the circulation while mast cells are in connective tissues and close to mucosal surfaces. When activated, these cells degranulate releasing pharmacological mediators. They play a prominent role in Type I hypersensitivity responses (IgE-mediated allergy) Histamine: vasodilation, vascular permeability cytokines: TNFα, IL-8, IL-5 attract neutrophils and eosinophils PAF (platelet activating factor) attracts basophils Mast cells have granules that contain powerful mediators of inflammation (e.g. histamine, cytokines) Have FcRs for IgE and can be triggered by specific allergens Mast cells reside in tissues, near blood vessel walls Basophils are somewhat similar to mast cells but are found in the circulation

Innate vs adaptive immunity

X. INNATE VERSUS ADAPTIVE IMMUNITY Having penetrated the external defenses, microbes come into contact with cells and products of the innate immune system. A number of cell types and defense molecules are usually present at the site of invasion or migrate to the site. This first line of defense is present at birth and changes little throughout the life of the individual. The cells and molecules of this innate system are mainly responsible for the first stages of expulsion of the microbe. This system works rapidly, gives rise to acute inflammation, and has some specificity for microbes. The adaptive immune system is brought into action while the innate immune system is trying to deal with the microbe, and especially if it is unable to eliminate it. The response of the adaptive immune system takes longer to develop, is highly specific for antigens associated with the microbe, and will remember that it has encountered the microbe (i.e. shows memory), which in turn leads to a more rapid expulsion of the microbe when it is subsequently encountered. The adaptive immune system is highly specific but unable to determine what is 'dangerous' and what is benign. It is the innate immune system that decides whether to attack a given microorganism or foreign antigen.

Knowing when to exit

adhesion molecules) that bind molecules on endothelial cells that line the blood vessel lumen. There are 'pairs' of interactors that are associated with entry into different tissues, e.g., mucosa, CNS. (A type of immunodeficiency is characterized by defects in molecules that target cells to the tissues: LAD. As attachment firms up and rolling stops, the interacting lymphocytes exit the blood and enter lymph nodes. Circulating lymphocytes have specific cell surface molecules that attach to specific surface 'addressins (L selectin ligand on naïve T cells) on blood vessel endothelial cells. These surface molecules permit lymphocytes to enter the lymph nodes via an area called the high endothelial venules (HEV). After entry and directed migration in the node (based on chemokines), if the T cells do not interact with MHC/peptide and become activated they leave the lymph nodes via efferent lymphatic vessels, eventually arriving in the thoracic duct, which empties back into the blood stream. Memory T cells are heterogeneous in their expression of adhesion molecules and chemokine receptors, thus providing access to different tissues. Other molecules called integrins (some common names are LFA-1, ICAM-1), which interact with endothelial cell surface molecules, are also part of the cascade of receptor events that firm up binding and allow exit from the blood. Both memory and naive T cells can recirculate through the blood. Lymphoid tissues are dynamic structures, wherein both T and B cells can access neo-antigen based on the movement of the lymphocytes, the antigen presenting cells, and the antigen. (The details of the B cell side of the story will be provided later.) Lymphocytes also traffic to the Mucosal Associated Lymphoid Tissue (MALT). One of the unique features of mucosal associated lymphoid tissues is that lymphocytes stimulated in one mucosal site can, in general, migrate to other mucosal sites to protect them from infection and disease. Thus, lymphocytes stimulated in the gut can migrate via the blood to other MALT sites such as the mammary glands and genitourinary tract, where their exit from the blood stream is mediated by specialized 'homing' (an older descriptive term) molecules.

TCR interactions

genetic recombination of TCR gene segments during early T cell development. The TCR is composed of an alpha chain and a beta chain. The process of T cell development occurs in the thymus. The thymus also deletes T cells that respond to self-antigens, resulting in a mature, peripheral T pool that is (by default) specific for foreign antigens. The hypervariable regions of the TCR (alpha and beta chains) interact directly with both MHC molecules and peptide antigens presented by MHC molecules. The TCR interacts with polymorphic regions of MHC molecules. Upon TCR binding to peptide:MHC, CD8 and CD4 co-receptors engage nonpolymorphic regions of MHC I and MHC II, respectively. Binding of the TCR to MHC:peptide induces T cell signaling through molecules on the T cell called CD3 and zeta-chain. This is called "Signal 1" and is crucial for T cell priming.

Type of hypersensitivity I reaction?

hay fever, food allergies, bronchial asthma, anaphylaxis

Th_2

interact with B cells and promote antibody production, make IL-4

Th_1

interact with macrophages and promote inflammation and development of CTLs, produce IFN-g

Th_17

involved in autoimmunity and inflammation

The rank order of deaths in the USA from allergies is due to:

medication>venom>food>latex

The sequence of events in Type I

sensitization phase: may require multiple exposures to elicit a clinical response effector phase: activation of mast cell (release of mediators) - immediate hypersensitivity reaction (minutes after repeat exposure to allergen) - late phase reaction (6-24 hours after repeat exposure to allergen)

Antigen

something that can react with antibodies (e.g. foreign pathogen or protein)

Immunogen

something that induces an immune response

Treg cells

suppress T cell function in the periphery and are important to prevent autoimmunity