Immune response to extracellular bacteria

The task of the immune system is to eliminate the bacteria and toxins.

The immune functions that are important for eliminating extracellular bacteria:

Complement

Monocyte/Macrophage

Neutrophil

B cell/antibodies

T cell/APCs

Complement

Origin: produced primarily by hepatocytes in liver. Activated macrophage can also produce it.

Location: blood and tissue fluid.

Function:

Lyse bacteriaOpsonize bacteria to facilitate phagocytosis

Induce inflammation

Clearance of antigen-antibody complex

Lysis of bacteriaComplements can be activated by three pathways.

1. Classical pathway:

Bacteria-antibody(Fc)-C1

C4, C2 C4b2aClassical C3convertase

C3 C3bB,D

C3bBb

Specificity

Alternative C3 convertase

C3 amplification loopC3b

C3b2Bb (alternative C5 convertase)

C4b2a3b (Classical C5 convertase)

C5 C5b C5b6789MAC

C6,7,8,9

Lysis of bacteria

IgM > IgG

Require activation of B cells.

Natural antibodies by B1 cells andCross-reacting antibodies may initiatethe reaction during primary immune response.

C-reactive protein (acute phase response) alsoactivate complement through C1.

BbC3b

degradation

RCA

C5b678, no C9

HRF, CD59

Lysis of bacteria

2. Lectin pathway:

Bacteria-MBL-MASP

C4, C2 C4b2aClassical C3convertase

C3 C3bB,D

C3bBb

Specificity

Alternative C3 convertase

C3 amplification loopC3b

C3b2Bb (alternative C5 convertase)

C4b2a3b (Classical C5 convertase)

C5 C5b C5b6789MAC

C6,7,8,9

Lysis of bacteria

Early response.

Upregulated during acute phase response.

C3b

degradation

RCABb

C5b678, no C9

HRF, CD59

Lysis of bacteria

3. Alternative pathway:

C3 C3bB,D

C3bBbAlternative C3 convertase

C3 amplification loopC3b

C3b2Bb (alternative C5 convertase)

C5 C5b C5b6789MAC

C6,7,8,9

Lysis of bacteria

Slow spontaneoushydrolysis

C3bBb

Degradation

RCAspecificity

HRF, CD59specificity

C5b678, no C9

Early response.

MAC is effective against Gram negative bacteria.

The thick peptidoglycan in the cell wall of GramPositive bacteria prevents the insertion of MAC.

Lysis of Bacteria

Opsonization

C3b is covalently attached to bacteria during complement activation.

Phagocytes (macrophages, neutrophils) have complement receptors that can recognize C3b.

Inflammation

C5a, C3aC4a

Mast cells

histamine

Prostaglandinleukotrine

vasodilaton(Increase blood flow)

inflammationvascular permeability

Leukocyte extravasation

(leakage of plasma)

(Recruitment of neutrophil and monocyte)

C3d facilitate the clustering of B cell coreceptor (CD21/CD19/TAPA1) with BCR and stimulate B cell activation.

Clearing of soluble antigen-antibody complex

Small antigen-antibody complexes (e.g. Toxin-antibody complex) are not phagocytosed.

Complement activation by the classical pathway

C3b covalently attached to the antigen

C3b-antigen-antibody complex is taken up by erythrocytes through CR1

Erythrocytes transport the complex to spleen and liver

The complex is transferred to Macrophages and internalized for degradation

Macrophage

Hematopoietic stem cell(HSC)

Myeloid pregenitor

Granulocyte-monocyte progenitor

monocyte

Bone marrow

Neutrophil1-6% 50-70%

Blood

Lymphoid progenitor

B cell progenitor

B cell

T cell progenitor

T cell

thymus

Lymphocyte (20-40%)

Primary lymphoid organ

Bone marrow

thymusLymphocyte maturation

Secondary lymphoid organ

Lymphocyte activation

Lymphocyte circulation

macrophage

Tissue

Function of Macrophage

1. Phagoctyosis

2. Produce inflammatory cytokines

3. Antigen presentation to T cells

4. Scavenger to eliminate apoptotic cells and immune complexes.

Phagocytosis

Bacteria (PAMPs)

Phagocytic receptors

Scavenger receptor

Mannose receptor

-glucan receptor

Opsoninreceptors

Fc receptor (antibody)

Complement receptor (C3b)

Collectin receptor (MBL)

Pentraxin receptor (C-reactive protein)

bacteria

phagosome

bacteria

phagolysosome

Bacterial killing

Oxygen-dependent

Oxygen-independent

Reactive oxygenintermediates

Reactive nitrogenintermediates

Degradedbacteria

exocytosis

Class II MHC

Antigen-presentation

Secretion of inflammatory mediatorsand antigen presentation

Bacteria (PAMPs) TLRs M activation

Inflammatory cytokines

IL-1

TNF-

IL-8

IL-6

Neutrophil activationAnd chemotaxis

Class II MHC

B7Antigen presentationT cell activation

Acute phase response(liver)

Local inflammationMBL

C-reactiveprotein

Phagocytosis

Prostaglandin, leukotriene inflammation

(Secondary lymphoid tissues)

Neutrophil

Hematopoietic stem cell(HSC)

Myeloid pregenitor

Granulocyte-monocyte progenitor

monocyte

Bone marrow

Neutrophil1-6% 50-70%

Blood

Lymphoid progenitor

B cell progenitor

B cell

T cell progenitor

T cell

thymus

Lymphocyte (20-40%)

Primary lymphoid organ

Bone marrow

thymusLymphocyte maturation

Secondary lymphoid organ

Lymphocyte activation

Lymphocyte circulation

macrophage

Tissue

Functions of NeutrophilsNeutrophils (blood circulation)

Rolling (mucin like CAM - E,P-selectin)

Activation by chemoattractants (IL8(CXCL8), C5a, N-formyl-peptides)

Activation of integrin (LFA-1, CR3, CR4, common subnit: CD18)

Stable interaction between LFA-1 with ICAM

Inflamed endothelial cells

Induced by inflammatorymediators

Chemotaxis by IL8, C5a, N-formyl-peptides, etc

Phagocytosis DegranulationKill bacteria

apoptosis Eliminated by macrophages

B cells

Hematopoietic stem cell(HSC)

Myeloid pregenitor

Granulocyte-monocyte progenitor

monocyte

Bone marrow

Neutrophil1-6% 50-70%

Blood

Lymphoid progenitor

B cell progenitor

B cell

T cell progenitor

T cell

thymus

Lymphocyte (20-40%)

Primary lymphoid organ

Bone marrow

thymusLymphocyte maturation

Secondary lymphoid organ

Lymphocyte activation

Lymphocyte circulation

macrophage

Tissue

B cell maturation

HSC CLPGL-ProB

DJ-ProB

LargePreB

SmallPreB

ImmatureB

sIgM+sIgM+

sIgD+

Mature B

B220 (CD45R)

CD19

C-kit

IL7R

SLC (5/VpreB)

RAG-1,2

TdT

IgH ()

IgL ()

Ig/

B cell marker

Surface marker (flow cytometry) V(D)J recombination

Bone marrow

2nd lymphoidtissues

B cell developmental stages are characterizedby surface markers and V(D)J recombination.

Cell surface marker is detected by Flow cytometry

Fluorescence-activated cell sorter (FACS)

V(D)J recombination

IgH : V-DJ joining

IgL ( and ): V-J joining

D-J joining

1. Organization of the IgH and IgL loci and the order of recombination.

2. Basic recombination mechanism.

JD

RAG-1,2 cleaves between RSS and the gene segments

JD

DNA repair to join the DNA breaks

D J +

Coding joint

Signal joint

12 97

heptamerspacernanomer

Recombination occurs between23-bp and 12-bp RSS (12-23 rule).RSS

Which parts correspond to the immunoglobulin V and C regions.

Imprecise coding junction (P nucleotide, N nucleotide)

23 79

V

V

V

Immunoglobulin diversity

1. Combinatorial diversity

Many combinations of V,D,J gene segments

Many combinations of heavy and light chains.

2. Junction imprecision

CDR3

Pre-BCR

Proliferation

Expand pre-Bpopultion

Shut off Rag1/2, TdT

Prevent V(D)J recombinationon the second allele

Activate transcriptionof k light chain

VJ recombination of locus.

Shut off SLC

Progression tothe next stageof maturation

5-6 roundsx 30-70

Btk: XLA

IgL rearrangement

1. Ordered rearrangement: -

3. Repeated rearrangement.

2. Allelic exclusion.

Negative selection

Multivalent self-antigen Clonal deletion

Soluble self-antigen AnergyBone marrow and periphery

Receptor editing (light chain) may rescue some self-reactive B cells in Bone marrow.

Alternative splicing vs polyA determines membranevs secreted Ig.

IgM is expressed first. IgD is expressed throughalternative splicing.

Ig diversity in chicken: gene conversion.

Lymphocyte RecirculationThe connection between lymphatic system and blood circulation.

B,T cellsBlood circulation

White pulpB cell follicles

T cell area

HEV (extravasation)

B cell follicles

T cell area

Blood circulation

Efferent lymphatic vessele

The circulation facilitatesthe encounter of lymphocyteswith antigens.

The major lymphoid tissues.

spleen

Other 2nd lympoid tissues

Antigen

M cell(MALT)

T cells

Hematopoietic stem cell(HSC)

Myeloid pregenitor

Granulocyte-monocyte progenitor

monocyte

Bone marrow

Neutrophil1-6% 50-70%

Blood

Lymphoid progenitor

B cell progenitor

B cell

T cell progenitor

T cell

thymus

Lymphocyte (20-40%)

Primary lymphoid organ

Bone marrow

thymusLymphocyte maturation

Secondary lymphoid organ

Lymphocyte activation

Lymphocyte circulation

macrophage

Tissue

T cell maturation

Double negative CD4-CD8-

Double positive CD4+CD8+

Single positive CD4+ or CD8+

RAG-1/2TdT

IL7

SCF

IL7R

c-kit

TCR loci

The structure and organization of TCR .

The order of rearrangement

Diversity

Combinatorial diversity (many combinations of gene segment and the two subunits)

Imprecise coding joint: CDR3

Commitment to or lineage

Pre-TCR signals the progression for further maturation along the lineage.

Proliferation

Expand + precursors

Degradation of RAG-2 and repression of RAG-1/2 expression

Prevent rearrangementOf the other allele (allelic exclusion)

Prevent rearrangement Of the and loci ( commitment)

Activation of CD4 and CD8 Expression(DP stage)

Transcriptional activationof TCR locus

Rearrangement of TCR

(100x)

-selection

(repeated rearrangementno allelic exclusion)

Positive selectionCD4, CD8 lineage commitment

CortexTCR+CD4+CD8+

cortex epithelial cells

Apoptosis Elimination by macrophage

TCR+CD4+ TCR+CD8+

Negative selection

None self-reactive Mature T cells

Apoptosis Elimination by macrophage

medullaDendritic cells and macrophages

Medulla epithelial cells (AIRE)

Lymphocyte RecirculationThe connection between lymphatic system and blood circulation.

B,T cellsBlood circulation

White pulpB cell follicles

T cell area

HEV (extravasation)

B cell follicles

T cell area

Blood circulation

Efferent lymphatic vessele

The circulation facilitatesthe encounter of lymphocyteswith antigens.

The major lymphoid tissues.

spleen

Other 2nd lympoid tissues

Antigen

M cell(MALT)

MHC and antigen presentation

Class I MHC: HLA-A,B,C (human); H-2D, L, K (mouse).

Class II MHC: HLA-DP, DQ, DR (human); H-2IA, IE (mouse).

Gene structure and diversity

Polymorphism and polygene allows the presentation of diverse peptides to T cells.

MHC interaction with TCR, CD4, CD8

TCR binds to Class I MHC-peptide complex.

TCR

T cell

Target cell

MHC

peptide

CDRs

CDR3 is the most variable part in TCR, and forms the major contact with the peptide.

CDR3 coincides with imprecisejunctions

CD4-Class II MHC, CD8-Class I MHC interaction stabilize TCR-MHC:peptide interaction and transmits activation signals.

Class II Class I

Activation signal

Antigen processing and presentation

EndocytosisPhagocytosismacropinocytosis

endosome

Dendritic cells

Macrophages

B cells

Thymus epithelial cells

APCs

All nucleated cells

8-10 amino acidPeptideHydrophobic C-terminusAnchor residue

13-18 amino acid peptide

TCR antigen may derive from internalpeptides while Ab eptitope is generallyon molecular surface.

Bacteria, toxin

virus

T cell activation

Antigen presenting cells:

Dendritic cells, macrophage, B cells

Antigen capture (type and location, migration)

Co-stimulatory molecule Distinction from non-antigen presenting cells

T cell activation T cell anergy

T cell activation:

TCR

CD4 or CD8clustering Phosphorylation

cascade

Lck, ZAP-70ITAM

Proliferation and differentiationInto effector cells

Co-stimuation (B7-CD28) stimulates IL-2 production.

B cell activation

BCR

Co-receptor(CD19,CD21,TAPA1)

clusteringPhosphorylationcascade

Lyn, Fyn, BlkSyk

BCRAg

Ag

C3d

endocytosis Class II MHC-peptide

B7

TH cell activation

Activated TH cells

CD40L

Cytokines(IL4, IL5)

CD40

Activated B cells

Plasma cells

Memory cells

Secret immunoglobulins

AntibodiesAntibody structure and function domains

CH1

CH2

CH3

VH VL

CL

Hinge region

Antigen binding site (CDR1,2,3)

Fab

Fc

Complementactivation Bind to Fc receptor

on phagocytes

Opsonin-mediated phagocytosis

Effector functions

IgH constant region determines antibody class.

The functions of antibodies

1. Neutralization of pathogen and toxin.

IgA is in external secretions (mucus, milk, tears, and saliva). IgA prevents the attachment of pathogen to mucous surface.

IgM also contributes to immunity in external secretions.

High affinity IgG is important for neutralizing toxins.

2. Oposonin for phagocytes. This is important for eliminating encapsulated bacteria.

IgG is the most effective.

3. Complement activation.

IgM > IgG

4. Maternal protection

IgG is transported across the placenta.

IgA in milk.

infection

10 2 3 4 5 6 7day

ComplementMast cellResident macrophage

NeutrophilsMonocytes Macrophage

Innate immunity

B and T cells

Adaptive immunity

Primary immune response

(immediate)

(after 5-7 days)

Secondary immune response

Innate immunity, Adaptive immunity

(within hours)

Present before infection

Limited and fixed specificity

Does not improve

Memory B and T cells

Develop in response to infection

Diverse and specific

Improve in specificity

Immunological memory

No memory