Week 3: Dendritic Cells

cDC deliver 3 signals to activate T cells

-Signal 1 is the Ag that T cells recognize through their TCRs in the context of MHC molecules. -Signal 2 are costimulatory molecules, like B7 (also called CD86) -Signal 3 are cytokines that the DCs produce to induce specific T cell differentiation (T helper 1, T follicular helper cells etc) -In absence of Signal 1, T cells remain resting -T cells that receive Signal 1 without Signal 2 do not receive the survival signal and they die.

Signals to DCs

Exogenous signal , coming from the pathogens (PAMPs, pathogen associated molecular patterns) -Endogenous signal, coming from stressed and damaged tissues (DAMPs, damage associated molecular patterns). -The encounter with PAMPs and/or DAMPs induce DC activation with their up-regulation of MHC and costimulatory molecules and the acquired ability to produce pro-inflammatory cytokines.

Self-Non Self Model of Immunity

-Innate immune cells have Pattern Recognition Receptors (PRRs) that bind conserved molecules shared by families of evolutionary distant organism like bacteria or viruses. -These molecules, called Pathogen Associated Molecular Patterns (PAMPs), are usually essentials for survival or pathogenicity of microorganisms; otherwise the microbes would lose them in order to escape from the immune system

Main Functions of the Innate Immune Cells

- Immunological Recognition through PRRs - Immune Effector function through phagocytosis and cytotoxicity, Fuel Inflammation - Antigen Presentation to Lymphocytes - Class regulation through production of cytokines - Tissue repair

TLR4 Signaling

-A major function of type I IFNs is to mediate, in an autocrine way, the activation of DCs induced by the triggering of TLRs. -As an example, here we have the signaling downstream of TLR-4: upon binding with the bacterial product LPS, a signaling cascade that utilizes MYD88 leads to NFkB activation and then production of several pro-inflammatory cytokines, while the branch that utilizes TRIF, leads to the activation of IRF3, the production of Type I IFNs that then in an autocrine manner stimulates the production of many Interferon Responsive Genes (IRG) including MHC and costimulatory molecules.

PRRs sense pathogens

-Innate immune system is activated through the Pattern Recognition Receptors that are triggered by Pathogen Associated Molecular Patterns and by Damage-Associated Molecular Patterns, and many call these PAMPs and DAMPs "danger signals", -TLR1, 2, 4, 5 and 6 are on the cell surface and recognize molecules conserved in extracellular bacteria -LPS from Gram-neg bacteria binds TLR4 -Lipoproteins from Gram-pos bacteria and fungi bind TLR2 and TLR6 -Flagellated bacteria bind TLR5. -TL3, 7, 8 and 9 are present in the endosomal compartment and recognize mostly nucleic acids like double stranded RNA (ligands for TLR3) and single stranded RNA (TLR7-8) and short sequences of DNA, CpGs (TLR-9) that can come from intracellular pathogens like viruses or DNA endocytosed from the extracellular space.

APCs are at the center of the immune response

-APCs, and Dendritic Cells (DCs), being the most powerful Ag Presenting Cells of the immune system, are at the center of the immune response. DCs can induce different immune responses or induce tolerance depending on the activatory or inhibitory signals and cytokine environment in which they are stimulated. -DCs can induce many T helper subsets: TH1 are T cells that help the CD8 to become killers and they also stimulate the microbicidal activity of macrophages during infections with intracellular pathogens; the TH2 help the activation of eosinophils and the production of IgE by B cells in response to parasites and allergens; TH17 cells help the clearance of extracellular bacteria activating neutrophils; follicular T helper cells help B cells to produce antibodies; also TH1 and TH2 can induce B cells to produce antibodies, leading the B cells to switch to different classes of Immunoglobulin (Ab) isotypes (igG1 vs. IgG2a for example), but the TFH have the right chemokine receptors (CXCR5) and costimulatory molecules (ICOS) to travel to the germinal centers and effectively help B cells; and many more T helper functions will be discovered by the next generation of scientists. -DCs also stimulate CD4 regulatory T cells, also called Treg, that are characterized by the expression of CD25 and of the transcription regulator FoxP-3 and their function is to suppress the immune response, as part of the maintenance of peripheral tolerance and at the conclusion of the immune response.

Conventional DCs mature through stages to become potent APCs

-Chemokines and chemokine receptors regulate where DCs go. -cDCs express CCR5 when they need to go to the peripheral tissues and be resident there. -Once the DCs have encountered an infection in the periphery, they start activating and they upregulate the chemokine receptor CCR7. -CCR7 allows DCs to move to the draining lymph node through the lymph and end up in the T cell area to present the phagocytosed Ags to T cells. -While traveling to the lymph nodes, DCs complete the process of activation, by up-regulating the MHC class I and MHC Class II molecules and the costimulatory molecules and adhesion molecules necessary to present the Ag to naïve T cells.

Cross-presentation over-come viral evasion mechanisms

-Cross-priming is the result of the cross-presentation: it is the activation of CD8+ T cells specific for Ags from apoptotic cells or other kind of particles that the dendritic cells have phagocytosed. -Cross-presenting DCs can cross-prime CD8+ T cells that are specific for intracellular pathogens that do not infect DCs. -Cross-presenting DCs can prime for intracellular pathogens, like some viruses, that infect DCs and inhibit their expression of MHC Class I or kill them.

Cross Presentation

-DCs are able to divert antigenic peptides from the endosomal compartment, where phagocytosed material is processed, to the cytosolic compartment from which is transferred to the endoplasmic reticulum, where the peptides are loaded on the MHC Class I molecules. -Cross-presentation is also important for the indirect presentation of Ags from the graft to CD8 T cells

Normal Presentation for Dendritic Cells

-DCs present on MHC class II peptides derived from material that they have phagocytosed; therefore, MHC Class II is the way for the DCs to activate the T helper cells against everything that is outside them, extracellular bacteria, immune complexes and also dead cells containing intracellular pathogens. -But the CD8+ T cells recognize their Ag in the context of MHC class I and in most cells MHC Class I displays peptides that derive from the cytosol of the same cell. -If these rules were rigid, DCs could present to CD8+ T cells only Ags coming from viruses that directly infected them, while they could not present Ags from viruses that for example infect epithelial cells, like the influenza virus.

Cross-talk between DC subsets is mediated by autocrine/paracrine Type I IFNs

-Different subsets of DCs produce different Type I IFNs. -myeloid DCs produce mostly IFNb while plasmacytoid DCs are the major producers of IFNa and this dialogue between subsets, that is so important for an effective response against viral infection, is unfortunately involved in the pathogenesis of some autoimmune diseases, like Systemic Lupus Erythematosus, in which excessive production of Type I IFNs and IFN Responsive genes has been found in PBMCs of SLE patients and Type I IFNs has been found to be pathogenic in murine models of lupus.

Cytokines: Signal 3 to induce T cell differentiation

-Each T cell subset is dependent on a specific cocktail of cytokines that induces the expression of specific transcription factors to differentiate and then each subset produces a specific cocktail of cytokines to perform its function. -Uncommitted T cells exposed to IL-12 and Interferon gamma (IFNg) develop into Th1 T cells that express the transcription factor t-bet and produce IL-2 and IFNg. -IL-4 induces GATA3 expression and differentiation in TH2 T cells that produce IL-4 and IL-5. -Our understanding of the effects of cytokines is complicated by overlapping conditions. Indeed, the combination of TGFb and IL-6 leads to expression of the trasncription factor RORgT and the differentiation of the pro-inflammatory TH17 T cells producing IL-17, while TGFb per se induces FoxP3 expression and the differentiation into regulatory T cells expressing TGFb and IL-10 and capable to inhibit the immune response.

Innate Immunity Function

-It recognizes when it is appropriate to mount an immune response: it is responsible for fighting the infections in the first few days from the encounter with a pathogen new for the immune system, when the adaptive immunity is not ready yet. -So, the innate immune cells recognize the infection and they also act as effector cells during the primary response. -Some of the innate cells, once they are activated, act also as Antigen Presenting cells (DCs, Macrophages and B cells), cells that are necessary to stimulate the T lymphocytes to activate and initiate the adaptive immunity.

Monocytes/Macrophages

-Monocytes are generated in the bone marrow, travel in the blood for about 8 hours and then migrate into the tissues during inflammation -In the tissues, there are also long-lived resident macrophages, which derive from precursors that left the bone marrow during the embryonic development. -Macrophages have different names depending on the tissue where they are resident: alveolar macrophages in the lung, histiocytes in the connective tissues, Kupffer cells in the liver, mesangial cells in the kidney, microglia in the brain, or osteoclasts in the bone.

More co-stimulatory molecules and CTLA-4

-Most costimulatory molecules belong to two families, the CD28/CD80-86 Ig superfamily and the TNF-TNFR family as shown in figure. CD40-CD40L interactions are bi-directional since CD40 signaling is pivotal in the licensing of DCs to activate CD8 CTLs and to give T cell help to B cells for antibody formation. -ICOS (Inducible Costimulatory molecule) is expressed on the cell surface of germinal center T cells and it is pivotal for the function of T follicular helper cells (Tfh), the CD4+ T cells required to help B cells. -Indeed, ICOS deficient mice have impaired germinal center formation and Ab isotype switching and absence of Tfh cells. -ICOS-ICOS-L interactions strengthen the T helper lineage and functions in all the subsets so far known, Th1, Th2, Th17 and Tfh. -ICOS-L is expressed by activated APCs. -Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) are two examples of inhibitory receptors of T cell activation. -These molecules are up-regulated on activated T cells 24-48 h after stimulation. -CTLA-4-deficient mice show activated peripheral T cells, splenomegaly, and diffuse lymphocytic infiltrates starting from one week of age. -CTLA-4 seems to inhibit T cell activation via at least two mechanisms: it directly stimulates phosphatases like SHP-1 that inhibit the signaling pathway downstream TCR. As a second mechanism, CTLA-4 competes for the binding of CD80-CD86 (once called B7.1 and B7.2) with CD28 and it is 20 times more avid for it. -A CTLA-4-Ig fusion protein (Abatacept, commercial name Orencia) is used in Rheumatoid Arthritis patients who failed to respond to TNF blockers. -The inhibitory molecule Programmed Death 1 (PD-1) is expressed on activated T and B cells and macrophages, suggesting that compared to CTLA-4, PD-1 may inhibit more immune responses. Upon association with the ligands PD-L1 (B7-H1) and PD-L2 (B7-DC), PD-1 binds SHP-1 and SHP-2 phosphatases and induces the dephosphorylation of proximal signaling molecules downstream of TCR. -The result is the decreased T-cell proliferation, survival and IL-2 production. -PD-1 is important in the termination of immune responses, induction of Tregs and maintenance of self tolerance. -PD-1-deficient mice develop a lupus-like disease by 6 months of age.

Dendritic Cells

-Most dendritic cells derive from bone marrow precursors and they travel through the blood to all the peripheral tissues where they reside in a resting state and act as the sentinel of the immune system, sampling the environment through phagocytosis and pinocytosis,waiting for a danger signal to active them. -Once they are activated, they travel to the draining lymph nodes where they present the Ag that they have acquired in the periphery, to stimulate the T cells and start an immune response. -T cells clonally expand and differentiate into effectors cells, then they leave the lymph node, they reach the site of inflammation. -A process less known is the one in which dendritic cells travel from the periphery to the lymph node to present in a tolerogenic manner self-Ag to establish and maintain peripheral tolerance of the self-reactive T cells that escaped negative selection in the thymus.

Monocyte/Macrophage functions -Phagocytosis? -Opsonization? -Antimicrobial? -Secretion of factors? -Antigen Presenting?

-Phagocytosis: ingestion, and digestion of microorganisms, injured or dead cells, cellular debris, and insoluble particles. Occurs through: chemotaxis, adherence to the pathogen or dead cell, formation of pseudopodia, phagosomes, and phagolysosomes; digestion of antigens through hydrolytic enzymes, H2O2, free radicals, lysozyme; exocytosis (elimination of digested contents). -Opsonization: facilitation of macrophage phagocytosis by antigen-antibody-(complement) complexes through specific macrophage receptors for the constant part of antibodies (CD16-CD32) and/or complement. -Antimicrobial activity: Macrophages produce factors able to kill microbes and eukaryotic cells too. They can be divided in 1) oxygen-dependent: reactive oxygen intermediates such as superoxide anion, hydroxyl radicals, H2O2, hypochlorite (long-lived oxidant) and nitric oxide (NO), nitrogen dioxide and nitrous acid. 2) oxygen-independent: lysozyme, defensins (circular, cysteine-rich peptides which form ion-permeable channels in bacterial and mammalian cell membranes). -Secretion of factors: IL-1, complement proteins, TNFa, IL-6, GM-CSF, M-CSF, G-CSF. Moreover, monocytes from the circulation extravasate into the site of infection and differentiate into "inflammatory monocytes", which can carry the above scavenging activities, and are also able to become Ag Presenting Cells: -Antigen processing and presentation - after phagocytosis, some digested antigenic peptides are bound to class II MHC antigens and presented on the macrophage surface for recognition by T cell receptors present on CD4+TH cells.

IFN-alpha/beta

-Plasmacytoid DCs are the major producers of large amounts of Type I IFNs, but these cytokines can be produced also by many other cell types during many type of inflammatory responses, upon endogenous as well as exogenous danger signals, via triggering of TLRs as well other danger receptors. -Their functions affect all the immune cells and most of the immune responses.

pDCs have distinct markers and functions

-Plasmacytoid DCs circulate in blood, bone marrow, and peripheral tissues and play a pivotal role in fighting viral infections by producing type I interferons (IFNs). -In human, pDCs are CD11c neg, CD123+ (IL-3R) and BDCA-2pos. -Initially, pDCs were considered to be poor antigen presenting cells but later it has been reported that pDCs can take up and present exogenous antigens derived from apoptotic cells, viruses and immune complexes. -Delivery of antigens to the C-type Lectin-like Receptors BDCA-2 and DCIR on human pDCs results in antigen presentation in vitro, although it inhibits pDCs production of type I IFNs. -It is possible to deliver in vivo in mice an Ag specifically to pDCs by injecting the Ag linked in a fusion protein with an Ab that binds the pDC-specific endocytic receptor -This leads to the activation of CD8+ T cells.

cDC uptake Ags to perform several types of Ag presentation

-Receptor mediated endocytosis of extracellular bacteria to present on MHC Class II/CD4 T cells -Macropinocytosis of bacteria or viruses to present on MHC Class II/CD4 T cells -Viral infection to present intracellular antigen to MHC Class I/CD8 T cells -Cross-presentation of exogenous viral antigens to present MHC Class I/CD8 T cell -Transfer of viral antigens from infected dendritic cell to resident dendritic cell presents to MHC Class I/CD8 T cell

Co-stimulatory molecules are engaged in the Immunological Synapse: CD80-86/CD28

-The CD28/CD80-CD86 was the first costimulatory pathway discovered. -CD28 is a Type I homodimeric 45-kD transmembrane glycoprotein constitutively expressed on T cells and is a member of the Ig superfamily, like its two cognate ligands, the costimulatory molecules CD80 (B7-1) and CD86 (B7-2). -These two costimulatory molecules are absent or expressed at very low levels on immature/resting DCs and are highly up-regulated upon activation/maturation. -Triggering of CD28 induces T cell proliferation, IL-2 production (via gene transcription and increased mRNA stability) and T cell survival. -When CD80/CD86 bind CD28, PI3K is activated and converts PIP2 to phosphatidylinositol trisphosphate (PIP3) at the plasma membrane, leading to the activation of the survival signaling pathway of Akt. -Akt promotes T cell survival by multiple mechanisms: it enhances the nuclear translocation of NF-κB, which induces pro-survival genes like Bcl-xl; it inhibits transcription factors that promote cell cycle arrest; it induces the transcription of NFAT-regulated IL-2. -CD28-deficient mice have depressed immune responses, indicating the importance of CD28 in T cell activation, but they are not completely deficient of adaptive immunity, indicating that costimulatory molecules other than CD80-CD86/CD28 can activate T cells. -In the immunological synapse, upon TCR sustained triggering, there is redistribution of the molecules important for T cell activation, with TCR-CD3-MHC-peptide in the center, together with costimulatory molecules, then around them adhesion molecules and finally in the periphery CD45-CD44 to maintain engaged the APC-T cell interaction

Danger Model of Immunity

-The Danger Model that says that Dendritic Cells sense danger. -They are activated by endogenous signals, coming from cells undergoing stress, damage or necrotic death. -These signals are called Damage Associated Molecular Patterns (DAMPs) and they bind the same PRR.

Adaptive Immune System

-The adaptive immunity is mediated by T and B lymphocytes that become fully operational only after birth and they require days to be ready to face the pathogens.

Dendritic Cell subsets: Migratory cDC

-The cDC main functions are antigen uptake and presentation -Migratory cDC, such as the Langerhans cells of the epidermis and the interstitial DCs of the dermis and other solid organs, that sample Ags in peripheral tissues, then migrate through the lymphatics into the lymph nodes, where they present the antigens to T cells. The CD103+ dendritic cells are also migratory and arrive to the draining LNs from the peripheral tissues and can present antigens both to induce immunity or tolerance, depending on their state of activation.

Innate Immune System

-The innate immunity is always ready to function, it is already operational at birth and it activates fast, in few hours, because is mediated by cascades of molecules that activate in matter of minutes. -These cascades include the complement, the antimicrobial peptides and the pro-inflammatory cytokines such as the Type I Interferons. -The innate immunity is mediated by cells like the phagocytes (granulocytes, the dendritic cells and the macrophages) and the Natural Killer cells, and they all activate in few hours.

cDC from peripheral tissues brings Ags to draining LNs

-The path of an APC presenting cells delivering Ags from the peripheral tissues to the draining LNs. -The DCs arrive through the afferent lymphatic vessels to the outer cortex in the T cell areas so they can directly present Ags to T cells or transfer their Ags to resident DCs.

PRRs sense tissue damage

-The same TLRs recognize also endogenous danger signals released from damaged tissues and dead cells. -TLR2, 4 and 6 recognize products of oxidative stress such as oxidized LDLs and oxidized phospholipids, products of tissue damage such as degradation products of the extracellular matrix, molecules released by dying cells like heat shock proteins and HMGB1. -The intracellular TL3, 7, 8 and 9 recognize self nucleic acids released by necrotic cells and mitochondrial DNA after they are shuttled to the endosomal compartment of APCs via endocytosis and phagocytosis.

Dendritic Cell subsets: Resident cDC

-There are cDCs resident in the lymphoid tissues and they derive from blood-borne precursors and acquire and present Ags within the lymphoid organs. -The tissue resident DCs can be divided into CD8+ and CD8- subtypes. -The CD8+cDC are CD11c+ CD8a+ CD205positive (also called DEC205) and are the major producers of IL-12p70 upon TLR and CD40L activation and they can initiate inflammatory Th1 responses. -They are also the best in up-taking apoptotic cells and cross-present exogenous antigens on MHC class I. -Thus, although they can activate CD4 T cells, they seem to be especially dedicated to activating CD8 CTLs. -The CD8-cDC (myeloid) are CD4+CD11b+ and are better in activating CD4 T cells.

Human DC subsets

-There are the Langerhans cells from the epidermis, the CD14+ interstitial DCs, the monocyte-derived DCs and the plasmacytoid DCs. -Human CD141+ DCs do not express CD8 but have all the functions of the CD8a+ DCs in mice. -They are in the blood and in secondary lymphoid organs such as tonsils and spleen. -They produce high levels of IL-12 and are efficient in cross-present exogenous antigens to CD8+ T-cells. -They express similar TLRs, other PRRs and adhesion molecules than the mouse CD8a+ DCs and also the same chemokine receptors.

cDCs have distinct markers and functions

-cDCs express CD11c and chemokine receptors, like CCR7. -They express MHC I and MHC class II. -In resting state, they express MHC Class II mostly in intracellular storages, and little to no costimulatory molecules. -Upon maturation/activation, they express MHC Class II on the plasmamembrane and up-regulate adhesion molecules like ICAM-1 and costimulatory molecules like CD80 and CD86 (new names for B7.1 and B7.2). -Conventional DCs can also express tissue-specific markers, depending on the tissue they are resident in: for example, langerin (CD207) is expressed by Langerhans cells (LC) and interstitial dermal DC localized in the skin; CD103+ is expressed by migrating DCs and DCs resident in the lamina propria (LP) of the small intestine; C-type lectin (DC-SIGN) is expressed by DCs present in the decidua and mucosal tissue; BDCA-1+ and BDCA-3+by DCs described in the lung.

Dendritic Cell subsets: pDC

-pDC are the major producers of type I interferons, a family of cytokines pivotal in the response against viral infections -pDCs can also present Ag after activation

pDCs are type I Interferon factories

-pDCs were originally discovered as minuscule population of cells in human blood traceable as interferon-producing cells because they are responsible for the strong production of type I interferons (IFNs) upon viral encounter. -They express TLR7 and TLR9 that allow them to sense viral nucleic acids within the early endosomes, triggering the antiviral immunity. -In few hours from the first encounter with a virus, pDCs convert up to 60% of their ER to transcribe type I IFN genes and produce up to 1000 times more IFNs than any other blood cell type. -Type I IFNs directly inhibit viral infection and also activate the antiviral functions of other cells, such as mDCs, NK cells, and B cells.

4 Main Tasks of the Immune System

1. It recognizes the presence and the severity of an infection. 2. It fights the infection during the effector phase 3. It contains the amplitude and regulates the type of immune response so to avoid harming the bystander tissue and then it stops the immune response when the infection has been eliminated. 4. Immunological memory develops, so it is more effective in fighting an infection the second time it encounters the same pathogen.