What is PBMC? 

All WBCs except granulocytes 

lymphocyte, monocyte, macrophage

What are white blood cells or leukocytes? 

Immune cells that carry out innate and adaptive immune repsonses. All are made in bone marrow, but have different destinations and functions. 

Innate: dendritic cells (in tissues, engulf antigens and present them)

macrophages (all tissues, engulf and degrade foreign particles, APC, monocyte in blood)

PMN (polymorphonuclear) in blood, granulocytes (NEB)

Mast cells (in blood, granulocyte, releases histamine)

Adaptive: in lymph nodes

mature dendritic cells present antigen to T cells

T-cells 

B-cells 

NK cells 

What cells are granulocytes? 

Contain granules in their cytoplasm. 

Includes neutrophils, eosinophils, basophils, and mast cells. 

What is PMN (polymorphonuclear leukocytes)?

Have irregularly shaped nuclei: neutrophils, eosinophils, basophils. 

What immune cells contain granules? 

Granulocytes: NEB and mast cells 

What immune cells are phagocytes? 

Monocytes, macrophages, all granulocytes, and immature dendritic cells. 

What cells are antigen presenting cells (APC)?

Dendritic cells, macrophages, B lymphocytes

Macrophages:

Liver: kupffer

Kidney: mesangial

Lung: alveolar macrophages

Brain: microglial cells 

What immune cells are innate? 

Neutrophils, eosinophils, basophils, mast cells, macrophages, immature dendritic cells 

What is adaptive immunity? 

Adaptive immunity consists of humoral (antibody mediated) and cell-mediated (cytotoxic T cells) and is the second step of immunity that is more specific to that pathogen. Adaptive is specific and has a memory, most cells are in the lymph nodes. 

What are the primary lymphoid tissues?

Primary/central lymphoid tissues refer to where the lymphocytes are generated, which is in bone marrow and thymus. 

All the cellular elements of the immune cells in blood arise from stem cells in which tissue? 

Bone marrow 

What is the consequence if there is no thymus? (nude mice) 

No thymus means no T cells, immune deficiency is present. 

What are the secondary lymphoid tissues?

Lymph nodes, spleen, and mucosal lymphoid tissues of the gut, nasal, and respiratory tract, urogenital tract and other mucosa. 

What immune cells are lymphoid lineages?

B cells, T cells, and NK cells 

What immune cells are myeloid lineages? 

Dendritic cells

Mast cells

Macrophages

Neurophils

Eosinophils

Basophils

(myeloid lineage comprises most of the innate immune system)

Where do circulating lymphocytes encounter antigens? 

In peripheral (secondary) lymphoid organs 

What are peyer's patches? 

These lie in the gut mucosa, have M cells (specialized epithelial cells to collect antigen)

Understand the microbial proteins that are produced by epithelial cells and phagocytes. 

Skin: fatty acids, β-defensins, cathelicidin, lamellar bodies

Gut: α-defensins (cryptdins), RegIII (lecticidins), cathelicidins

Lungs: α-defensins , cathelicidins

ENT: β-defensins, histidins

Macrophages: superoxide, cathlicidin, macrophage elastase-derived peptide  

What is a lysozyme? 

Lysozymes have specificity for certain tissue in the GI tract; they can directly cleave peptidoglycans to expose cell walls, and they're able to destroy bacteria. 

 What is a defensin?

A defensin is a small protein that interrupts the lipid bilayer and forms a pore in cell wall, release antimicrobial peptides. Activated by proteolysis

What is a paneth cell? 

These are in the intestine, and they secrete defensins. 

What is a PAMP? 

Examples of PAMP

Pathogen associated microbial patterns.

Ie Gram-positive bacteria has a signature pattern of peptidoglycans on its surface, gram-negative bacteria contains lipopolysaccharides on its surface. 

What is PRR? 

Examples of PRR 

PRR are pattern recognition receptors, macrophages express a number of receptors that allow them to recognize different pathogens.

For example, MBL: mannose-binding lectin, TLR: toll-like receptors, NLRs: NOD-like receptors, and NOD: nucleotide-binding 

Scavenger receptor and MBL which have CRB (carb recognition domain) 

ROS, Nitric oxide, lysozymes, defensins, superoxide, hydrogen peroxide, acids. 

Rapid release of ROS from cells, Caused by a transient increase in oxygen consumption during the production of microbicidal oxygen metabolites. Neutrophils engulf and kill microbes to which they bind, bacterial peptides activate Rac2, and bacteria are taken up into phagosomes. Phagosomes fuse with granules inside, Rac2 induces assembly of functional NADPH oxidase, get generation of O2-, super oxide. From super oxide you get hydrogen peroxide, then radicals from there.

NADPH oxidase converts oxygen molecules to superoxide O₂^-, this is the respiratory burst. Formed as a result of chemokines. 

What causes Chronic Granulomatous Disease? 

Patients with mutations in the genes for components of NADPH oxidase have macrophages and neutrophils that are unable to initiate a respiratory burst. They are then susceptible to chronic bacterial infection, and then chronic inflammation, from granulomas in the tissues. 

What can be found in purulent exudates (pus)? 

Dead and dying neutrophils and dying bacteria. 

  What are the structure domains for TLR?

Extracellular LRR domain, intracellular TIR domain with signaling box. 

What is the ligand for TLR4?

LPS (gram-negative bacteria), and lipoteichoic acids (gram-positive bacteria) 

What is the ligand for TLR9?

DNA with unmethylated CpG, in bacteria and herpesviruses 

What transcription factors can be activated upon engaging TLR signaling?

What is NF-kB? 

NF-kB is in the cytoplasm as an inactive heterotrimer composed of 2 Rel family proteins and inhibitory IkB. You get degradation of IkB to release NfkB, then NFkB is allowed to move to nucleus to activate responsive genes. 

What is NLR or caterpillar? 

Examples of NLR. 

NLD is a NOD-like receptor, examples are the CARD subfamily, the Pyrin subfamily and the BIR subfamily.

Their structure includes a lRR domain for ligand sensing and autoregulation, a NOD domain for nucleotide binding and self-oligomerization, and an N-terminal effector domain for signal transduction and activation of the inflammatory response. 

Understand NALP3 inflammasome

NALP3 signaling activates caspase-1 cleavage, which leads to IL-1β and IL-18

Why does the production of mature cytokine IL-1β and IL-18 require NALP3 inflammasome?

Because it needs to be cleaved (activated) by caspase 1. 

What is RIG-1-like helicase (RLH)? 

Example of RLH

RIG1 recognizes RNA, initiates a signal cascade, able to generate an active form of NF-kB or IRF-3

What are the signs of acute inflammation? 

Heat, redness, pain, swelling, loss of function

What cytokines do activated macrophages produce? 

IL-Iβ, TNF-α, IL-6, CXCL8, IL-12              

Which cytokines are produced by activated macrophages are involved in fever elevation? 

IL-Iβ, TNF-α, IL-6 (endogenous pyrogens, which elevate body temperature), which then induce acute-phase protein production in the liver 

What is acute phase protein? Where is acute phase protein produced? 

C-reactive protein, MBL: surfactant proteins SP-A and SP-D. Produced in the liver. 

What cytokines induce the production of acute phase protein? 

Examples of acute phase protein? 

Surfactant proteins SP-A and SP-D 

What is the function of acute phase protein? 

Activation of complement opsonization, which induces phagocytosis. 

How can aspirin or ibuprofen reduce a fever? 

They are non-selective inhibitors of the COX enzymes, and therefore stop inflammatory cascade. COX enzymes lead to prostaglandins and thromboxanes, which lead to fever and pain. By inhibiting COX enzymes, they stop part of this cascade to stop PG and Tx production. 

What is the function of the coagulation system? 

To form a cross-linked fibrin polymer to stop blood loss, and to prevent the spread of the pathogen via the blood. 

What are chemokines? 

Chemokines promote chemotaxis in direction of the highest concentration. 

   Example of chemokine receptor on monocytes and chemokine produced by endothelial cells (to attaract monocytes to the inflamed tissues)

On monocytes: CXCL8

Produced by endothelial cells: CXCL8

1. CX3C

2. ELR CXC and non-ELR CXC

3. 4C CC

4. 6C CC 

Neutrophils 

What is the mechanism that leukocytes transit the bloodstream? 

Extravasation:

rolling adhesion
tight adhesion
diapedesis
migration

CAMs are proteins located on the cell surface involved with the binding with leukocytes, other cells or with the ECM in the process called cell adhesion.

Families:

Ig (immunoglobulin)

Superfamily (IgSF CAMs)

Integrins

Musin-like

Selectins 

 Involved in extravasation. 

Examples of selectins 

Which cells produce E-selectins? 

Activated endothelium

TNF-α induces the rapid externalization of granules in endothelial cells, the Weibel-Palade bodies, which contain performed P-selectin, expressed within minutes on surface of endothelial cells. E-selectin is synthesized shortly after. 

Both interact with s-Le^x

Examples of integrins 

α1;β2, slide 33 of 44 (lecture 3)

(CAMs, allow immune cells to bind epithelial cells and exit blood stream)

Selectin binding slows down leukocyte, Integrins are stronger, bind second because their expression needs to be induced. Integrin binding is firm and stronger, allows leukocyte to adhere to blood vessels to allow transmigration into tissue side. 

Mainly expressed by leukocytes 

Chronic inflammation: 

signs and explanation? 

Infiltration with mononuclear cells (macrophages, lymphocytes, plasma cells)

Tissue destruction- done by inflammatory cells

Repair involving angiogenesis and fibrosis

Leukocyte adhesion deficiency? Defect in beta2 chain, recurrent bacterial infections and impaired healing of wounds. 

Large number of different plasma proteins that interact with another both to opsonize pathogens and to induce inflammatory response.

Functions of complement:

1. MAC-induced cell lysis

2. Complement fragment induced changes in

            Vascular Permeability: C3a and C5a acting as anaphylatoxins stimulate histamine release from mast cells

            Mediator release: C5a stimulate additional inflammatory mediator release

            Chemotaxis: C5a is a powerful chemoattractant

            Opsinization: C3b acts as an opsinin

Where can we find complements? 

On pathogen surfaces and antigen surfaces 

Why does fetal bovine serum (FBS) need to be heated to 56ºC inactivated before using in cell cultures? 

C4 à C4a and C4b

C2à C2a and C2b

C3 à C3a and C3b 

C1 is first. 
C1q, C1r, and C1s complex

C1q globublins bind directly to pathogen surface, C-reactive protein (IL-6), and Fc portion of antibody.

Binding causes conformational change in C1r/C1s. Enzymatic activity of C1r release C1s. C1s is serine protease. C1s finds and cleaves C4.  

MBL or ficolin binds carbohydrate on pathogen. 

Lectin-like domain binds mannose

Fibrinogen-like domain binds N-acylglucosamine

MASP domains have enzymatic activity: MASP 1 unknown function, MASP 2 serine protease. 

MASP 2 cleaves C4, then like classical pathway from there on. 

What is the C3 convertase from the alternative pathway? 

Which complement component is opsonin? 

C3b- binds to complement receptors (CR1, CD35) on phagocytic cells to promote phagocytosis. 

What are the major functions of complement-mediated stimulation of inflammation? 

1. Opsonize particles for phagocytosis (need to reach C3b stage)

2. Elicit inflammatory reaction by acting on leukocytes, mast cells, endothelium

3. Complement-mediated cytolysis

(Vascular permeability, mediator release, chemotaxis, opsonization)

It can initiate opsonization, initiating phagocytosis. 

What is Serpin (C1 inhibitor)?

Chronic spontaneous complement activation leads to production of excess cleaved fragments of C4 and C3. Complex cascade of events with kinin and complement cascades results in increased vascular permeability and tissue edema. 

Complement regulatory proteins interact with C3b if C3bBb forms on the surface of host cells, preventing the formation of C3 convertase.

         DAF or CD55, delay-accelerating factor

         CR1, complement receptor

         MCP or CD46, membrane cofactor of proteolysis

          Factor H 

Which protein prevents final assembly of the MAC? 

CD59 prevents final assembly of the MAC at the C8 to C9 stage

(also, vitronectin S protein binds soluble C5b67 and prevents insertion of the MAC into the cell membrane)

How can activated macrophages contribute to inflammatory responses? 

Signaling causes the secretion of pro-inflammatory cytokines (IL-1beta, IL-6, TNFa), chemokines (chemoattractant cytokines), chemical mediators, and co-stimulatory molecules, which all contribute to local inflammatory responses. 

Examples of cytokines that are produced by activated macrophages? 

Examples of acute-phase proteins that are produced by hepatocytes? 

SP-A, SP-D, MBL, Fibrinogen, C-reactive protein, serum amyloid protein

Which acute phase proteins can activate complement?

C-reactive protein, MBL

Examples of surface receptors of macrophages: 

Fc receptor

Complement receptor

Cytokine receptor

Chemokine receptor

Hormone receptor

Lipoprotein lipid receptor

ECM (fibronectin, Laminin, collagen) receptor 

Surface molecule of macrophage, an Integrin:  CR3, CD11b/CD18, α_M:β2

Name signaling pathways that are involved in macrophage activation: 

TLR

TNF-α receptor

Cytokine receptor (JAK STAT)

Chemokine receptor (GPCR)

Interferes with viral replication, induces Mx proteins, 2’-5’ linked adenosine oligomers, and the kinase PKR. Increases MHC class I expression and antigen presentation in all cells, activated dendritic cells and macrophages, activates NK cells to kill virus-infected cells, induces chemokines to recruit lymphocytes. 

Type I: IFNα and IFNβ

Type II: IFNγ

TLR-3

(signals via TRIF to induce IFN gene expression)

(TLR-7 binds ssRNA, signals via MyD88 to induce IFN gene expression)

Cytoplasmic replication of virus produces uncapped RNA with a 5’-triphosphate, Viral RNA binding to RIG-1 induces RIG-1 to associate with adaptor MAVS, dimerization. MAVS induces production of inflammatory cytokines and IFNs via NFkB and IRFs

Which transcription factors are activated upon TLR-3 signaling pathway? 

 Why can interferons control viral infection? 

Which type of dendritic cells (DCs) can make large amount of interferons? 

pDC (plasmacytoid)

For viral infection these make large amounts of IFNs and Interferon producing cells.

NOT for activating naive T Cells (conventional DCs)

Name the STAT protein that is activated upon IFNγ stimulation? 

STAT1

1) Macrophages activated in the liver and spleen secrete TNF-alpha into the bloodstream

2) Systemic edema, decreased blood volume, collapse of vessels

3) Disseminated intravascular coagulation leads to wasting and multiple organ failure because of over-production of cytokines and stimulatory fragments 

4) Death 

What is MyD88?

Is the TLR2 signaling pathway MyD88-dependent? 

Lymphocyte that has characteristic surface antigens. It releases lytic granules that kill virus-infected cells. 

Two types:

1) Cytokine producers

2) Killers

Name common surface markers for identifying human NK cell population. 

CD3-negative CD56+

Name cytokines that can activate NK cells 

IFN-α, IFN-β, IL-12

Activated NK cells can secrete what cytokines? 

IFNγ, TNF, GM-CSF

What are the mechanisms responsible for NK cell cytotoxicity? 

Directed exocytosis of secretory lysosomes that contain lytic proteins including perforins, granzymes, and the Fas ligand. 

What can be found in the granule of NK cells that functions to induce apoptosis? 

Granzyme B

Why don't NK cells kill normal host cells? 

Normal host cells express MHC Class I, which provides a negative signal to NK cells. When MHC Class I are altered/absent, no negative signal is received to inhibit the signals, only positive signals are received, the NK cell is triggered to release its granule contents, inducing apoptosis. 

NK cells express both activating and inhibitory receptors. Why do the inhibitory receptors convey inhibitory signals? 

How can activating receptors convey activating signals? 

Through up-regulation of gene transcription of cytokine production, granule production, etc. 

What are ITAM and ITIM?

ITAM= immunoreceptor tyrosine-based activation motifs

ITIM= immunoreceptor tyrosine-based inhibitory motifs 

Name the families of NK receptors 

KIR (killer cell immunoglobulin-like receptors)

NCR (natural cytotoxicity receptor)

KLR (killer-like receptor)

Name the two major kinases used in the NK cell activating receptors. 

PI3-kinase, ERK 

What is ADCC? 

Antibody dependent Cell-mediated cytotoxicity 

Give examples of NK activating and inhibitory receptors

Activating: KIR2DS, ECRly, CD16, NGK2D, DAP10

Inhibitory: KIR2DL, KIR3DL, CD94/NKG2A 

What are the ligands for NKG2D?

MHC-like molecules: MIC-A, MIC-B or RAET1 family members (induced by cellular stress)

How can you use NK cells to eliminate target tumor cells? 

Altering the surface receptors of NK cells can create a mismatch to the surface markers of the cancer cells, marking them for destruction. 

Give examples of innate-like lymphocytes 

B-1 cells: Make natural antibody, protect against infection with streptococcus pneumoniae. Ligands aren't MHC-associated, can't be boosted.

Epithelial γ:δ cells: produce cytokines rapidly. Ligands are MHC class IB associated, can't be boosted.

NK T cells: produce cytokines rapidly, ligands are lipids bound to CD1d, can't be boosted