immunology prelim 2

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cellular immunity

T cell activities

humoral immunity

activities of antibodies

Plasma cells

-make antibodies
-sessile in the BM or medulla of lymph node (IgM)
-they are the most important part of B cell development

Plasmablasts

-the most immature blood cell that is considered a plasma cell instead of a B cell
-migrates from LN to BM, but can be diverted to sites of inflammation where it will release antibodies

Where does B cell activation take place?

Sec. lymphoid organs

Three signals for B cells

1. BCR and co-receptor complex bind antigens.
2. Co-stimulatory receptor is CD40
3. Cytokines

Cross linking surface Ig

-Signal communicated to interior by Ig-alpha, Ig-Beta, which each contain two ITAMs
-Src kinases or protein tyrosine kinases are activated by the binding and phosphorylate the chains at ITAMs
-Tyrosine kinase Syk binds Ig-beta that allows assembly of large signaling complex

B cell co-repceptor includes

-CD21
-CD19 (complement receptor)
-CD81 (TAPA-1)

Co-receptors purpose

-enhances activation

CR2 or CD21

-complement receptor 2
-in complement process, C3b fragments on pathogen
-C3b is the ligand for the complement CR1 receptor on B cells which facilitates its cleavage by iC3b and C3d
-CR1, in doing this, increases B co-receptors on pathogen
-CR2 binds C3d, which brings the cell closer to pathogen

CD19

-after CR2 binds, brings CD19 close to tyrosine kinase Lyn, which phosphorylates it
-intracellular signal begins

Conjugation of C3d

Improves antibody responses by a lot

What are signals 2 and 3 of B cell activation*

-B cell serves as APC to activated T cell
-CD40 = signal 2
-cytokines = signal 3
*to activate T cell or to an already activated T cell?

How long do B and T cells remain associated?

10 min - 1 hr

B and T cells recognize the same antigenic site. T/F

Not usually - B cells will present many epitopes to T cells

Where does B cell develop?

Bone marrow, blood and sec. lymphoid tissue, lymphoid follicles, antigen

Bone marrow

Unrestricted repertoire of immature B cells. Tolerance induction

Blood and sec. lymphoid tissue

Self-tolerant mature B cels and anergized B cells. Additional tolerance induction -> then enter lymphoid follicles for antigen activation

Traffic pattern naïve B cells in LN

-CCL21 and 19 attract through HEV into LN
-attract to primary follicle where stimulated by dendritic cells and cytokines

Where do B cells circulate through?

Lymph, blood, sec. lymphoid tissue

Two pathways determine how develop:

-B cells move to medullary cords to become IgM
-B cells may more to germinal centers to become other

Important events in germinal center

-Clonal expansion
-Class switching
-Somatic hypermutation
-Differentiation of memory cells or plasma cells

Clonal expansion

-In dark zone
-B cells become known as centroblasts
-Clonally expand

FDC's with antigen

-One's that fix complement more immunogenic and stronger B cell stimulation

Iccosomes

Immune complex coated bodies - have antigen all over surface for B cell binding

Somatic hypermutation

-During rapid proliferation
-CDR1, 2, 3 of V domain are hotspots
-High rate of mutation 10^-3 base pair/division
-precise
-Involves AID

AID

-Activation indued cytidine deaminase (AID)

Affinity

-The attraction between an antigen and an antibody
-Improves with time
-# mutations on heavy and light chain increases

Class switching

-In light zone under influence of T cells and specific cytokines
-Recombination of constant genes in heavy chain locus

Switch sites

-Sites targeted by AID
-Upstream of each CH except C delta

No switch with IgD

-No switch b/ no switch region upstream of C delta

What is class switch driven by?

-T cell cytokines

IL-4

IgG1, IgE

IL-5

Augments IgA production

IFN-gamma

IgG3, IgG2a

TGF-B

IgG2b, IgA

Plasma cells

-No surface Ig
-2000 ab/second
-Live for yrs. in BM
-400,000 in BM
-Continuous exposure to CD40L inhibits differentiation to plasma
-IL-10 promotes it

B cell vs. Plasma cell

-B cell have surface Ig, MHC and can go through processes in germinal center
-Plasma cells just secrete

IL-10

Leads to plasma cell

IL-4

Leads to memory cells

Memory cells

-Recirculate and live long
-High affinity receptors
-More likely than naïve cells to differentiate into plasma cells for homeostatic reasons
-Crucial to vaccination's effects

Antiserum

-Blood serum containing polyclonal antibodies

Complement system

-Bordet discovered antiserum could lyse bacteria
-Adding normal serum to antiserum complemented effects
-Heat response

Activation of complement system

Involves a series of proteolytic cleavages that yields products that can serve as opsonins, chemotactic factors, cellular activators, and lytic agents

C'

-Both innate and adaptive immunity
-Dangerous and highly regulated

Complement can lead to macular degeneration

...

All complement things involve

C3 cleaved to C3a and C3b and C3b binding to surface of pathogen

C3b

-Opsonizes particles for engulfment by macrophages, which have receptors for it

C3a

-Chemotactic for neutrophils and macrophages

Cleavage of C3 exposes:

-Thioester bond
-Which can be soluble to H20 or bound to pathogen through R-OH or R-NH2
-After made in liver, bond available in plasma for pathogen

Opsonization

-Process whereby pathogen is marked for ingestion and destruction by a phagocyte
-CR1 on macrophage recognizes C3b on pathogen - endocytosis, makes vesicle, fuses with lysosome to form phagolysosome

Alternative pathway

-Pathogen surface creates local environment conducive to activate complement

Lectin pathway

-Mannose-binding lectin binds to pathogen surface to activate complement

Classical pathway

-C-reactive proteins or antibody binds to specific antigen on pathogen surface (adaptive immunity driving innate)

Alternative pathway steps

-C3 bound to H20 spontaneously in plasma to become iC3
-binds factor B
-protease factor D cleaves B to Bb, with Ba released
-Now, iC3Bb
-Intact C3 binds iC3Bb which cleaves it to form C3a, C3b

Then once C3b is bound...

-Factor B binds, D cleaves to make C3bBb, C3 makes many more C3b's on pathogen surface

Limits on complement activation on surface of animal cells

-Inhibitory proteins H and I
-H binds to sialic acid and makes sure that if C3b deposited, that it cleaved by I to make iC3b
-Bacteria don't have sialic acid so no limits

Factor I

-Limits animal cell C3b
-Also can cleave iC3b to C3d, which is recognized by CR2 on FDC and B lymphocytes

Antibodys' effects on Activating complement

-IgM low affinity, very good with complement
-IgG3 also very good
-IgG1 and 1 chosen in therapeutic antibody design depends on desired outcome

C1

-binds C-reactive protein which binds phosphocholine on pathogen
-or binds IgM

Specific IgM conformation exposes C1q binding sites - staple conformation

...

Once C3b is bound will generate more convertase via alt. pathway

C4b assoc. with C2a
-becomes a convertase for C3

Process of classical

1. C1 binds ab/ag complex
2. Deposition of C4b by C1
3. Deposition of C3b by C4b2a
4. Deposition of C3b by C3bBb

Pentameric IgM vs. IgG

C1 binds single staple
C1q binds multiple IgG

Mannose binding lectin

-MBL and its associated serene proteases mimics C1q
-Has 2 MASP1 and 2

The process of lectin binding

-Once MBL binds pathogen, activates MASP2 which cleaves C4 to C4a/b; also C2 to C2a/b
-C4b binds to microbial surface
-C2a binds surface of C4b forming classical C3 convertase
-Binds C3 and forms C3b

Terminal pathway

-C3b2Bb can cleave C5 to C5b, which leads to terminal pathway
-Binds C6, 7, 8, 9 on surface

C5a

-very important chemotactic factor
*** what does that diagram mean???

Osteoarthritis

-C5 activation a cause
-Eculizumab

Acute phase response

-Triggered in the liver by cytokines (TNF-a, IL-1B, and IL6) generated in innate response
-Generates additional mediators of inflammation to promote destruction of infectious agents
-Can be prolonged if infection not cleared

Effector cells

Macrophages and mast cells

Pro-inflamatory cytokines

-Released by macrophages
-IL-6, TNG-a, IL-1B, CXCL8, IL-12
-What should we know about the pro-inflammatory responses?

IL-1, 6/ TNF-a

Act on liver, bone marrow, hypothalamus, fat/muscle --> activation of complement opsonization, phagocytosis, decreased viral and bacterial replication

IL-6

-Acts on hepatocytes to induce acute phase response
-then, C-reactive protein binds phosphocholine on bacterial surfaces, acting as an opsonin and as a complement activator
-Mannose-binding lectin binds to carbs on bacterial surfaces -> complement activator

There are mechanical, anti-microbial and chemical defenses

...

Pathogens vary in associations with hosts...

Extracellular, intracellular

Disease

-Caused by direct and indirect effects of pathogens: a great deal of what we see as disease during infections is the result of the immune response

Signs of inflammation

-Swelling, redness, heat, pain, loss of function

Defensins

-Amphipathic
-Positively charged peptides that associate with negatively charged urfaces of bacteria and then create pores in the bacteria to kill it
-Attack bacteria, viruses, and fungi
-35 to 40 amino acids
-Ancient

alpha-defensins

-Expressed mainly by neutrophils and paneth cells

beta-defensins

-Expressed by a large range of cells

Paneth cells

-Located at the crypts of the small intestine
-HD5, HD6

Myeloid cells

-Cells that are induced - require a cellular response to a threat or pathogen

Recognition of pathogens

PRR, PAMP, TLR
-outcomes are phagocytosis and cytokine production

TLR

-Family of signaling receptors which is specific for a different set of microbial products
-Important in pathogen recognition
-Activate innate cells and indirectly influence adaptive immunity

Know structure of TLR

-N and C
-Leucine-rich repeats
-Pathogen recognition domain
-TIR domain
-Transmembrane polypeptide with a TLR

What to know from second slide on 10?

...

Bacteria outer surface

Lippolysarccharide, then outer membrane, then peptidoglycan, then inner membrane

Lectins

-Receptors and plasma proteins that recognize carbohydrates

Scavenger receptor

-Phagocytic receptor of macrophages that is not a lectin

TLR4

Important in body's defense against gram negative bacteria
-recognize bacterial lipopolysaccharide with MD2, LPS and CD14 at cell surface`

NFkB

-After TLR with gram negative bacteria
-Transcription factor for inflammatory cytokines

The process

-TLR4, MD2, CD14, LPS on macrophage surface
-MyD88 binds TLR4 and activates IRAK4 to phosphorylate TRAF6 to phosphorylate IKK
-IKK phosphorylates IkB leading to release of NFkB
-NFkB goes to nucleus to make inflammatory cytokines

Alternative pathway

-After detects ligands, TLR4 or TLR3, bind TRIF and TRAM
-Phosphorylate TRAF3
-Kinase cascade
-Phosphorylate IRF3, which directs transcription ---IFN a, b

TNF-a

-Local inflammatory response that helps contain infections
-Also systemic effects

CXCL8

-Local only - helps attract neutrophils to site

IL-1, IL-6, TNFa

-All critical role in inducing acute phase response
-All systemic effects

IL-12

-Local only - Activates NK cells

IL-6

-Systemic only - acute phase response; fever

TNFa

-Increase release of plasma proteins into tissue
-Increased phagocyte and lymphocyte migration into tissue through having endothelial cells increase expression of adhesion molecules
-Increased platelet adhesion to blood vessel wall

Septic shock ** explain

-When systemic infection with gram negative bacteria
-TNF-a into bloodstream
-Decreased blood volume, hypopoteinemia, neutropenia, decreased blood volume causes collapse of vessels
-Dissemnated intravascular coagulation leads to wasting and multiple organ failure

Phagocytosis

1. Bacterium becomes attached to membrane evagination called pseudopodia
2. Ingested forming phagosome
3. Fuses with lysosome
4. Digested and digestion products released from the cell

Where are TLRs?

-Dendritic cells
-TLR engagement activates DC to upregulate CCR7 which binds CCL21 in sec. lymph tissue to move to lymph node
-Also upregulates MHC and B7 to activate DC

What happens when injury in epithelia?

-Tissue damage causes release of vasoactivate and chemotactic factors to increase blood flow and permeability
-Allows cells and fluid to go there
-Phagocytosis

Bradykinin, Fibrin degradation, Plamin and complement activation

...

Mast cells

-Important initiators of adaptive response
-Reside near blood vessels in connective tissue; produce same chemo/cytokine
-Macrophages with granules
-Degranulate in response to IgE bound to surface receptors
-have TLRs and receptors for C3a, C5a
-Activated by bacteria and compement
-

Mast cells

-Break down membrane phospholipids to generate vasoactive and chemo-attractive molecules

Lyso-PAF

-From mast cells breaking down phospholipids
-Can cause clotting

Arachidonic acid

-From mast cells breaking down phospholipids
-Vascular permeability and neutrophil chemotaxis

Aspirin

-Gets in the way of the cyclooxygenase pathways

IL-8

A chemoattractant for neutrophils
-Made by macrophages

neutrophils

-High number in blood and large reserve in BM
-roll stop drop mechanism

How do neutrophils work?

-Bacterium is phagocytosed by neutrophil
-Fuses with azurophilic (lysozyme, defensins, myeloperoxidase, and proteases) and specific granules (lysozyme, NADPH)
-pH rises, antimicrobial response activated
-pH drops and allows lysosome to bind to phagosome and completely destroy bacteria
-Neutrophil dies and phagocytosed by macrophage

Respiratory burst

-Neutrophils have an enzymatic pathway toxic to micro-organisms
-WIth Superoxide and H2O2
-Key feature of neutrophils

Leishmania

-Infected the ear
-Leishmania red and green neutrophils
-Neutrophils aren't necessarily directed at micro-organisms

Virus infection

-Cell mediated immunity is necessary for clearance of a primary infection, antibodies are crucial to preventing secondary infections
-IFN-gamma, CTLs, NK, macrophage

Influenza has an antigenic hemaglutinin

...

Capsin

-Encapsules genome of virus
-For influenza, single stranded RNA (ssRNA)

Innate defense against viruses

-TLRs that recognize ssRNA which will initiate innate response to the virus
-TLR7, 8

TLR 7 and 8

-In endosome can bind ssRNA -> signal that leads to production of IFN-a, b

If virus infects via endosome, or endocytosed by DC, viral RNA can bind TLRs

...

Interferon and viruses

-Viruses bind surface of cells, release nucleic acids bound by TLR in cell -> IFN responses and transcription
-IFN-B released by cell and binds another cell to prevent viral infection of that cell
-also IFN B bind same cell to prompt IFN A
-paracrine and autocrine responses

IFN-A, B

-cause cell to stop synthesizing protein
-inhibits viral replication

Second slide of 5?

...

Type I interferons

-alpha and beta
-three functions are induce resistance to viral replication by activating cellular genes that destroy viral mRNA and inhibit the translation of viral proteins
-Increase expression of ligands for NK cell receptors on virus infected cels
-Active NK cells to kill virus infected cells

NK cells

-Lymphocytes of innate immunity that specialize in defense against viral infections
-Lymphoid progenitor
-"large granular lymphocytes"
-produce IFN-gamma when stimulated by IL-12 and TNF-alpha
-cytotoxic which is promoted by exposure to IFNa/B or IL-12

NK cell receptors

-most are extracellular ligand binding regions of Ig domains or lectin like domains

Activating NK receptors

-Short cytoplasmic tails
-Changed amino acid residues

Inhibitory NK receptors

-Long cytoplasmic tails that contain short amino acid sequence called immuno-receptor tyrosine based inhibitory motif ITIM
-binds protein pho sphatases that act to inhibit

NK cells distinguish cells

-NKG2D- an activating receptor present on all human NK cells
-ligands for NKG2D are MIC-A and MIC-B, which are only expressed by viral or traumatic cells
-healthy cells only have interaction b/w inhibitory receptor and ligand whereas viral cells have NKG2D interact with MIC to activate and over-rule inhibitory response

Time until virus gone

1. Proudciton of IFN a, B, TNFa, and IL12
2. NK cells
3. T cell killing

Movement of effector cells

-Dendritic cells pick up bacterial antigens in skin and move to draining lymphatic vessel and settle in T cell areas of LN

Antigen-specific T cell movement

-Trapped in LN where activated briefly then enter blood and diverted to sites of infection by altered endothelial cell surface receptors

Cell-surface molecules and T cells

-VLA-4 binds VCAM on endothelial T cells in blood vessels at sites of infection to extravascate into sites of inflammation
-T cells don't have L-selectin - can't cross HEV - rather enter LN via afferent lymphatics

Broadly defined effector functions

-Effector cells do not need co-stimulatory receptors to engage their target cells

CD8+ T cells activation/function

-On professional APC
-Proliterate by IL-2
-Targets any somatic cell

CTL

-Cytotxic T cell
-Recognizes virus-infected cell
-Programs cell to die and moves to another cell

Proteins in granules of cytotoxic T cells

-Perforin, granzymes, granulysin

Perforin

-Aids in delivering contents of granules in cytoplasm of target cell

Granzymes

-Serine proteases
-Activates apoptosis once in cytoplasm of target cell

Granulysin

-Antimicrobial and induces apoptosis

CTL function

-Collision and non-specific adhesion doesn't work
-Engagement of the TCR allows polarization of T cell and release of lytic granules

What happens when a naïve CD8 binds class I plus non-professional APC?

Anergy

CD8+ cytokines

-IFN-gamma, LTa, TNFa

CD4+ activation

-Activated by TCR and C4 binding
-CD28 with B7
-IL-6, IL-4, IL-12, TGF-B cytokines from APC
-Cytokine signal directs differentiation

IL-6

-Acts on lymphocytes, enhancing responses

IL-12

-Acts on Naive T cells and makes them Th1

Different cytokine differentiation on T cell

-TGF-B then FoxP3 then Treg
-IL-6 TFH
-TFG-B, IL-6 TH17 cells
-IL-12, IFN-gamma then Tbet and IL2 then Th1
-IL-4 then GATA3 IL4 IL5 then Th2

IL2 and IL4

Important for T cell growth and development

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