202 terms


60% of circulating leukocytes
half life of 8 hours
4% circulating leukocytes
Differentiate into macrophages when enter tissues
-More effective phagocytes than monocytes
-APC for CD4+ T cell activation in secondary immune response - at site of infection
- MCP 1 - chemoattractant for monocytes/macrophages
- IL-8 - chemoattractant for neutrophils
- IL-1 and TNF - auto/paracrine activation of macrophages
- IL-6 - acts in an endocrine manner to tell liver to release CRP, an opsonin
-IL-12 - Activates NK cells and causes them to secrete IFNg-increased cytotoxicity of macrophages (iNOS).
-The IFNg also induces differentiation of Thp to Th1 cells

- Express CD4 and CCR5 and CXCR4 - HIV infection
Toll-like receptors
- Are membrane-bound (TLR) pattern recognition receptors (PRR) on phagocytes (macrophages and neutrophils) and dendritic cells
- transmembrane
- Recognize PAMPs (Pathogen associated molecular pattern)
- Trigger cascades of biochemical events that lead to secretion of inflammatory cytokines and other innate immune responses
- 10 that recognize a different patterns
Nod-like receptors
Nod1 and Nod2 (NLRs)
Mutations in Nod2 lead to Crohn's Disease
Target pathogen to be phagocytosed
- Complement activation - C3b (interacting with CR-1)
- B cell activation - IgG (FcgR, CD 32, CD 16)
- Cytokine mediated activation of hepatocytes - CRP/IL-6 (CRP-BS)
Degradation of antigens in phagosomes
1. Lysosomal enzymes -
- lactoferrin - binds iron
- lysozyme - destroys muramic acid in bacterial cell walls
- defensins - permeabilize bacterial and fungal membranes
- myeloperoxidase - generates hypochlorite with H2O2 and chloride ion (ROI) - halogenating cell walls

2. ROI - NADPH Oxidase - parts are found in membrane and cytosol and assembled after phagocytosis. Glucose to PPP to make NADPH. Oxidase uses O2 and NADPH to generate superoxide anion (ROI) - initiating step for other ROIs. Respiratory burst follows (esp. imp. in neutrophils)

3. RNI - NO lipid and water soluble and cytotoxic. More potent when NO reacts with ROIs to make RNIs. Esp. imp. for mycobacteria, IC bacteria, Leishmania which are resistant to ROIs. Target ETC to inhibit respiratory cycle. NO also damages phagocytes as well
but has short half life.
Regulation of iNOS
-Catalyzes arginine and O2 to citrulline and NO
-Requires IFNg (in addition to phagocytosis) to be activated. (maybe TNF as well)
- Downregulated by IL-10, IL-4, TGF b (most effective) (from activated T cells)
In both circulation and tissues
half life 8-10 hours
Main defense against parasites, esp. helminth worms
Requires IgE generated in adaptive immunity
Have FceR that binds IgE abs that are bound to worms
Interaction triggers degranulation, releasing major basic protein (MBP - disrupts parasite membranes) and eosinophilic cationic protein (ECP - forms pores in membrane)
Mast cells also crosslinks with IgE with their own FceRs, releasing histamine.
Natural Killer Cells
aka. large granular lymphocytes (LGLs)
- Found in blood, spleen, peritoneal exudate
- protection against tumor production and early stages of viral infection
- recognize and kil infected or malignantly transformed cells
- KAR - recognition of viral peptides embedded in virally infected cell surface - kill by receptor mediated toxicity
- KIR - recognition of self, healthy cells, interacts with MHC class I plus self - ignore cell.
- Stress, viral infections, tumors decrease expression of Class I plus self peptides (more susceptible to NK cell, but escape immune surveillance by adaptive immunity)
- Indirect/Ab dependent cell mediated toxicity - NK cells express FcgR III (CD 16) that interacts with IgG. Cell killed
- Kills by releasing granzyme B and perforin. Perforin (in presence of Ca++) inserts itself into cell membrane and allows granzyme B to go inside, which activates caspases that induce apoptosis (not lysis)
- Secrete IFNg upon activation of induction by IL-12 and IL-15 or IL-18, which protects surrounding cells from viral attack and and enhances T cell activity
- enhanced killing abilities with IFNg and IL-2 (T cells) and IL-12 (macrophages and dendritic cells)
-Become LAKs with IL-2 - enhanced cytotoxic activity - tumor treatment
Mast cells and Basophils
Receptor-ligand interactions
- FceR with IgE - crosslinkage in order for degranulation to occur
- C5aR with C5a - anaphylatoxin, chemotactic for neutrophils
- C4aR + C3aR with C3a or C4a - anaphylatoxin

Cytokines produced
- IL3 - mast cell proliferation
- TNF, MIP-Ia - inflammation
- IL4, IL-13 - IgE production, mucus secretion
- IL-5 - acts on bone marrow - eosinophil production and activation

Mediators released
- pre-formed in granules: histamine, enzymes (eg. tryptase)
- lipid mediators produced on activation - platelet activating factor, leukotrienes (C4, D4, E4 = SRS-A) - same effect as histamine but slow, PGD2
Antigen presenting cells
B cells, dendritic cells, macrophages
Require on their surface: MHC II and co-stimulatory molecules such as B7 (CD80 + CD86) and CD40 -- increased with Ag capture or by cytokine signalling
- MHC II ONLY on APCs while MHC I on ALL nucleated cells (so APCs have both)

Cells that present to CD8+ T cells are termed target cells because they are targets for cell-mediated death
Dendritic Cells
-Most efficient Ag presenter during primary immune response
- Immature DC are ubiquitous (except brain)
- Gatekeepers/sentinels - capture Ags and carry them to secondary lymphoid tissues
-Immature DCs express a lot of FcgR (making them phagocytes initially) and little MHC II and co-stimulatory molecules
- Mature as they migrate, allowing T cells to recognize Ag bound to it
- Express CD4 - determines T cell lineage. CD4 as well as CCR5 and/or CXCR4 are receptors for HIV-1
B Cells
- Recognize Ag via Ag specific receptors (mIg)
- Highly specific -- bind Ag at low concentrations -- predominant APC when Ag is limiting

For T dependent antigens the B cell acts as an APC:
-The receptor and antigen are internalised into an endosomal vesicle.
-Processing of the material involves its breakdown following fusion with lysosomes.
-The receptors are recycled to the surface.
-The foreign peptides then encounter MHC class II antigens in a 'chimeric' endosome, following a second fusion.
MHC loci
Humans (HLA - chrom. 6):
Class I -- B, C, A
Class II -- DP, DQ, DR

Mice (H-2 - chrom. 17):
Class I -- D, L, K
Class II -- I-A, I-E
MHC Class II
- Heterodimer with 2 transmembrane polymorphic polypeptide chains (a and b)
- a1 and b1 contribute to peptide binding cleft which can hold bigger peptides (15-30 aa) because it is open at both ends
- antigens interact with a co-receptor molecule, the CD4 (contains D1-D4 domains with long cytoplasmic tail - signal transduction) present on helper T cell populations.
- The interaction is with the β2 domain of the constant part of Class II
- Class II molecules bind peptides which are from exogenous sources, generated within intracellular acidifed endosomes.
MHC Class I
-Heavy chain non-covalently associated with b2-macroglobulin (non-polymorphic)
-No transmembrane domain
- b2M - no Ag binding, but needed for proper I folding
- This peptide binding cleft capable of binding peptides of around 8 -10 amino acids length (cleft closed at both ends) and is made up of a1 and a2 domains
- Class I molecules interact with the CD8 molecule of cytotoxic T-cells
- CD8 interacts with an invariant part of the class I molecule, situated in the 3 domain.
- bind peptides only from endogenous sources
- co-dominant expression - 6 different class I molecules possible
Presentation to T helper cells
1. Uptake of Ag by APC
2. Fusion of endocytic vesicle with lysosomes that release their contents into the endosome
3. Ag broken down in the acidified endosome and smIg recycled
4. 2nd fusion with an endosome that contains MHC II (from ER) - chimeric endosome
5. Ii degraded and Ag peptide is allowed to bind in MHC II groove
6. Chimeric endosome migrates and fuses with cell membrane
7. Ag is presented on cell membrane bound to MHC II
8. T cell R recognizes and in presence of co-stim. molecules, T cell is activated to secrete cytokines
Presentation to cytotoxic T cells
1. Endogenous Ag is degraded via a proteosome into smaller fragments (targeted by ubiquitin attachment)
2. In ER, new Class I is attached to calnexin (chaperone - partially folded state), which dissociates after b2M binds and associates with TAP-1 (and TAP-2)
3. TAP allows endogenous peptides to be actively transported inside ER
4. binding of a suitable peptide to the partially folded MHC Class I molecule allows it to be released from the TAP.
5. The Class I MHC: peptide complex is directed to the Golgi Complex, for transportation to the cell surface, where the complex is expressed
Cross presentation
-Exogenous Ags presented by Class I on dendritic cells to CD8+ T cells -- cross-priming
- Ag escapes endosome to cytosol and are processed via the endogenous Ag pathway (proteosome)
-Rickettsia is an example - rapidly lyse the endosome before phagolysosome forms
Lipid and glycolipid antigen presentation
- presented on CD1 (CD1a-CD1e) molecules, which are structurally similar to class I MHC -they form a complex with β2M (endogenous).
- The processing and presentation of the CD1-antigen complex is similar to that described for class II molecules (exogenous)
-Different members are expressed on a range of cell types BUT dendritic cells (main presenter) possess all members of this family.
-The CD1 molecules possess a group of hydrophobic amino acids which forms a hydrophobic pocket, at which the lipid/glycolipid antigen can interact.
-Presentation is to NKT cells - characteristics of both NK and T cells
- mycobacterial infections
Antigen binding site
On variable region, which contains hypervariable region or complementarity determining region (CDR) - in both light and heavy chains. Forms the paratope - part of variable region that contacts the epitopes on the Ag
6 hypervariable regions - far in primary structure but close in tertiary structure to produce binding site
Note: framework region are the sequences with little variability
Rodney Porter and antibody digestion
Papain - 2 Fab + 1 Fc
Pepsin - (Fab)2 + bits of Fc (Fab held together with S-S bond)
Structure: monomer, dimer, or timer
-held together by a J chain
Half-life: 1 week
Subtype: IgA1 and IgA2
Location: MALT, intravascular-little in circulation-useless, secretions, epithelia (except skin), major Ab of milk and colostrum
Function: 1st line defense - most imp. Ig in external secretions; protection of mucosa - immunosurveillance - bind orgs before they bind M cells; activates alternative pathway of complement; breast-feeding - mother's immunity passed down
1. Mucosa IgA secreted as dimeric IgA by plasma cells in lamina propria.
2. Dimeric IgA binds secretory component (poly Ig), which protects Ig from harsh environment (enzymes/acid)
3. Both are endocytosed and transported to lumenal surface
4. Secretory component is cleaved with IgA keeping a part of the fragment
5. IgA released into intestinal lumen - called SIgA
Structure: exist only in membrane bound form on naive B cells along with IgM - not secreted (both have same variable regions and recognize same Ag)
Location: low serum concentration
Function: No protective role as soluble Ag; unknown; low serum levels reflects B cell death
Structure: monomeric
Location: Barely detectable in serum; bound to FceR on mast cells and basophils
Half-life: 2 days
Function: Multivalent Ag binding to IgE (bound to mast cells and basophils via FceR) results in crosslinking of FceRs leading to degranulation (release of inflammatory and vasoactive mediators); protection against helminth infection (can interact with FceR on eosinophils); reaginic
Structure: monomer; 20 allotypic forms (Gm); 4 subtypes (different in constant region)
Subtypes: IgG1 highest and IgG4 lowest; IgG3 half life only 1 week, but most potent complement activator; IgG4 no activation of complement of binding to FcgR
Location: 75% circulating Ag; circulates btwn blood and interstitial fluid
Half-life: 3 weeks (long - Ig remains elevated for longer periods in secondary response)
Function: transported across placenta - mother's immunity passed to child for 4 months; facilitiation of opsonization (Fc with Ag interacts with FcgR of phagocytes); ADCC by NK cells; complement activation (3>1>2, not 4); neutralization of viruses and toxins (Ag can't bind to cell surface); weak agglutination of cells (not good enough structure); major Ab produced in secondary response - isotype switching; in breast milk (not as much as IgA)
Structure: membrane bound is monomeric; secreted is pentameric held together by J chain
Location: 15% of all Ig; intravascular; only isotype expressed on immature B cells; expressed on naive B cells with IgD
Half-life: 5-7 days
Function: 10 Ag binding sites - high avidity (although low affinity) for Ag; best activator of complement - requires only one IgM Ag/Ag complex; 1st Ab produced in all primary responses; excellent agglutinator (isohemogglutinins of RBCs)
Rh antigens
Expressed on RBCs of 85% population
During placental bleed or during birthing, baby's Rh+ in contact with mother Rh-. She develops antibodies and IgG can cross placenta and attack baby's RBCs - erythroblastosis fetalis or hemolytic disease of the newborn
ABO Blood System and Isohemogluttinins
Naturally occuring Abs to the blood groups (Ag) you lack - from gut flora (newborns dont have them)
•A Blood Group
-Cell surface antigen A
-Isohaemagglutinin in plasma Anti B (IgM)
•B Blood Group
-Cell surface antigen B
-Isohaemagglutinin in plasma Anti A (IgM)
•O Blood Group
-Cell surface antigen -neither A nor B
-Isohaemagglutinin in plasma Anti A and Anti B
•AB Blood Group
-Cell surface antigens A and B
-Isohaemagglutinin in plasma ~ None.
Cells possessing FcgR
B cells - inhibits adaptive humoral response
Function of complement
1. facilitate interaction of phagocytes to induce opsinin-mediated phagocytosis
2. induce osmotic lysis of microbes
3. enhance vascular permeability by inducing degranulation of mast cells and basophils
4. induce chemotaxis of neutrophils
5. facilitate immune complex elimination
Classical pathway of complement
Ab-dependent (IgG -12,3 and IgM).
Need either a single IgM or 2 adjacent IgG
C1 - C1q (Fc binding), C1r, C1s (esterase)
1. Combination with Ag exposes C1 binding site on Fc of Ig
2. C1 becomes attached (complement fixation)
3. Interaction of C1 with Fcg/Fcm activates serine protease activity of C1s
4. generation of convertases - active C1 cleaves C4 to C4a and C4b
5. C4a is an anaphylotoxin (inflammation)
6. C4b onto microbial surfaces
7. C1 then cleaves C2 to C2a and C2b (C2b - weak kinin-vascular permeability/inflammation)
8. C2a attaches to Ag bound C4b to produce classical C3 convertase - C4b2a
9. C3 convertase splits C3 to C3a and C3b (multiple molecules of C3 are converted to produce an amplification loop)
10. C3a is another anaphylatoxin
11. C3b attaches to the microbial surface and acts as an opsonin, aids in the elimination of immune complexes.
12. C3b also attaches to the C3 Convertase to produce the classical C5 Convertase C4b2a3b.
13. C5 is cleaved C5a + C5b
14. C5a is an anaphylatoxin and causes mast cell or basophil degranulation (vascular permeability), chemotactic for phagocytes, so recruits these cells to the site of infection.
15. C5b attaches C6 and C7 which becomes attached onto the target cell membrane
16. C8 and multiple C9 (up to 16) molecules attach forming the Membrane Attack complex (MAC)
•The multiple C9 molecules assemble to form a pore of around 100Å diameter.
•These pores are large enough to allow the escape of small molecules and electrolytes down concentration gradients.
•The larger molecules remain within the target cell, increasing the osmotic pressure.
•Water enters the cell by osmosis and the cell swells and ruptures ~ osmotic lysis.
Alternative pathway of complement
Innate (older) mechanism (no Ab involvement) so no C1, C2, C4.
1. Spontaneous generation of C3b from circulating C3 (C3 tickover).
2. In absence of infection, C3b is inactivated
3. C3b binds to microbes (mostly bacteria) and prevented from binding to self by regulatory proteins. Can act as an opsonin
4. C3b binds Factor B
5. Cleaved by Factor D to Ba and Bb
6. Ba is soluble and diffuses away (no clear function). Bb binds to the surface bound C3b to produce C3bBb
7. C3bBb is stabilised by the addition of the next component Properdin (P) - resulting complex C3bPBb is the alternative C3 convertase.
8. This complex cleaves many more C3 molecules (amplification loop)
9. Some of the C3b acts as an opsonin the rest attaches to the C3 convertase to produce the Alternative C5 convertase C3bPBb3b.
10. C5 is cleaved C5a + C5b
11. C5a is an anaphylatoxin and causes mast cell or basophil degranulation (vascular permeability), chemotactic for phagocytes, so recruits these cells to the site of infection.
12. C5b attaches C6 and C7 which becomes attached onto the target cell membrane
13. C8 and multiple C9 (up to 16) molecules attach forming the Membrane Attack complex (MAC)
C1INH (C1 esterase inhibitor)
Regulation of classical pathway
-Dissociates C1r and C1s from the complex - no activation of classical pathway (no splitting of C4 and C2)
-inactivates kallikrein
Decay Accelerating Factor (DAF)
Regulation of both classical and alternative pathways
-binds membrane bound C4b and C3b, blocking the formation of C3 convertase (both). If already formed, DAF competitively binds with C3b or C4b promoting dissociation
- Displaces Bb from C3b (alternative)
Regulation of classical pathway
-Displaces C2a from the complex (binds and competes)
-binds to fluid phase C4b preventing attachment to cells
-Competitively binds C4b promoting dissociatin of C3 convertase
-cofactor for Factor I
Factor I
Regulates both pathways
- Factor I cleaves C4b to inactive forms, needs cofactor C4bp (C4 binding protein) - inhibiting classical C3 convertase
- Complement receptor 1 (CR1) cleaves C3b to yield inactive C3bi
Anaphylatoxin Inhibitor (AI)
Regulation of both pathways
Inhibits binding of C3a, C4a, and C5a to receptors on mast cells and basophils
Factor H
Regulation of alternative pathway
Binds C3b, displacing Bb, acts as cofactor for Factor I - inhibits C3 convertase
S-Protein (Vitronectin)
Inhibiting the insertion of C7 into the membrane, hence inhibits MAC formation
Homologous Restriction Factor (HRF)
Binds to C8 preventing formation of MAC
CD59 (Protectin)
CD59/HRF20 bind to C8 preventing formation of MAC
Links complement and coagulation pathways
1. Tissue damage converts XII to XIIa
2. XIIa activates pre-k to kallikrein
3. Kallikrein:
- activates more XIIa
- activates C5 to C5a and C5b
- converts kininogen to bradykinin (vasodilator)
- activates PMNLs
Complement Receptors
-Present on phagocytes, B cells, erythrocytes
-Binds C3b, C3bi and C4b
- opsonin mediated recognition of Ag, clearance of immune complexes
-Present on B cells
-Binds C3bi
-Mode of EBV infection
-Present on mast cells and basophils
-Binds C3a and C4a
-Present on mast cells and basophils
-Binds C5a
-vascular permeability
B cell receptor complex (BCR)
mIg, CD79a/CD79b (Iga-Igb) (which couples mIg to intracellular signaling pathways - signal transduction)
CD19, CD21, CD81
mIg variable chain in B cells
Light chain variable region - V and J segments
Heavy chain variable region - V, D, J segments
Avidity vs. Affinity
Forces between Ag and Ag binding site are non-covalent
Attraction between the two is affinity
Overall attraction is avidity
Unique variable regions due to
1. Multiple copies of germline V, D, J gene segments
2. Random selection and combination of V, D, J segments - somatic recombination
3. Junctional diversity generated by the addition or deletion of bases
4. Random assortment of light and heavy chains
5. Somatic mutation/affinity maturation (NOT in T cells) will fine tune the BCR
Somatic Recombination
Occurs in bone marrow during B cell development and in Thymus cortex during T cell development
-DNA in the loci that encode the variable regions (V, D, J) is cut and recombined to make an intact gene for the variable regions of the light and heavy chains
- V, D, J are present as multiple germline genes - are recombined
- Triggered by RAG-1 and RAG-2 (recombination activating genes) genes which bind to RSS motif (signals gene seg. start)
- Light chain (B) or alpha chain (T) - one J and V to form VJ
- Heavy (B) or beta (T)- one D and J to get DJ and then one V to get VDJ
- random selection is called combinatorial diversity
- junctional diversity - Tdt (terminal deoxynucleotidyl transferase) incorporates nucleotides at junctions to keep an open reading frame
Allelic Exclusion
Successful rearrangement of a heavy chain variable region or beta chain variable region from one chromosome inhibits the somatic recombination of the heavy chain/beta chain variable region on the other member of the chromosome pair
--all mIg present on the surface of a B cell/T cell will have the same heavy/beta chain variable region to give binding specificity (On B cells, 2 different constant regions though - m and d)
-- if both alleles are recombined, cell dies
--25,000 TCRs identical variable regions on cell
Stages of B cell development
Occurs in the bone marrow
1. Pro-B cell
2. Pre-B cell
3. Immature B cell
4. Mature B cell
Pro-B cell stage
Initial Transcription of
•Recombination activating genes RAG-1 and RAG-2
•Tdt (terminal deoxynucleotide transferase)
•Pseudo light chain
•CD19 (Pan B cell marker)
Pre-B cell stage
-Somatic Recombination of V gene heavy chain (m) and allelic exclusion.
-Initial transcription of heavy chain (V + mC)
-Association of 2 rearranged heavy chains m with pseudo light chains making the pre-BCR
-Expression of pre-BCR with CD79a/CD79b
-Somatic Recombination of light chain V genes and allelic exclusion -- occurs AFTER heavy chain produced
-CD20 expression (from late pre-B cell onwards)
-Late pre-B cells are CD19+CD20+.
Immature B cell stage
-Initial transcription of k or l light chain.
-Association of k or l chains with to form the BCR.
-IgM BCR expressed along with CD79a/CD79b.
-Down regulation of pseudo light chain
-Down regulation of pre-BCR
-Tolerance induction (negative selection). Cells with autoreactive BCR are made to undergo apoptosis or are inactivated (induction of anergy or non responsiveness)
Mature B cell stage
-Alternative Splicing of hnRNA
•V region is spliced with chain C region.
•Surface expression of IgM and IgD (both have identical V regions due to allelic exclusion)
-Surface expression of adhesion molecules (L-selectin) which allow interaction with vascular addressin molecules on high endothelial venules (HEV).
-Surface expression of CD40 (binds to CD154 on T cells, acts as co-stimulatory molecule)

•Naïve B cells migrate from the marrow and enter the blood stream.
•They populate B cell areas of peripheral lymphoid tissues.
•If they fail to encounter antigen in the 2°lymphoid organ they recirculate (lymphocyte trafficking).
•Exit from the blood into lymph nodes involves interaction between L-Selectin and vascular addressins on HEV.
•Entry into the spleen occurs at terminal branches of the arterioles (open circulation) at the white pulp.
B cell exit from blood
•Naïve B cells migrate from the marrow and enter the blood stream.
•They populate B cell areas of peripheral lymphoid tissues.
•If they fail to encounter antigen in the 2°lymphoid organ they recirculate (lymphocyte trafficking).
•Exit from the blood into lymph nodes involves interaction between L-Selectin and vascular addressins on HEV.
•Entry into the spleen occurs at terminal branches of the arterioles (open circulation) at the white pulp.
Immune response depends on Ag entry
Primary immune response occurs in secondary lymphoid tissues
Ag entry in:
blood -- spleen (white pulp)
mucosal tissues -- MALT (M cells)
tissues -- lymph nodes

same for B and T cell activation
Activation of naive B cells
-Occurs in outer areas of follicles where T cell help is not limiting (B cells in follicles, T cells surround follicles)
•Antigen induced crosslinking of BCR complex (T cell dependent - occurs in interfollicular regions)
•Endocytosis into cytosolic endosome.
•Lysosomes fuse with the endosome degredation of antigen by proteases.
•Fusion and formation of chimeric vesicle
-Endosome containing antigenic peptides
-Endosome containing MHC Class II molecule
•Formation of MHC Class II-Antigen peptide complex.
•Translocated to the B cell surface.
•Presented to TCR of activated CD4+ T helper cell (Th2)
•Conjugate formed further stabilised by adhesion molecules e.g. ICAM-1/LFA-1 and LFA-3/CD2.
•Several other B cell co-stimulatory signals generated due to interactions between B7(CD80/CD86)/CD28 and CD40/CD40L (CD154)
•Activated naïve B cells undergo clonal expansion (selective)
•Following the initial burst of proliferation activated B cells and T cells migrate to 1°follicles.
•Enhanced proliferation of B cells leads to germinal centre formation.
•The germinal centre is the characteristic of a 2°follicle.
Germinal Center Events in 1°Response
-B cell clonal expansion.
-Isotype switching (switch recombination).
-Affinity maturation (somatic mutation) the affinity of the Ab generated increases with time following Ag exposure.
-Differentiation to plasma cells ~ secreting large amounts of Ab.
-Differentiation to a population of memory cells ~ capable of rapid differentiation on second or subsequent exposure.
•CD40 and CD40L interaction delivers an essential stimulatory signal.
•CD40L (CD154) is expressed on activated Th2cells.
Plasma Cell differentiation
•B cells posses mIg whilst plasma cells secrete antibodies.
•Secreted antibodies lack the C terminus cytoplasmic and transmembrane domains which hold the Ig in the B cell membrane.
•The secreted Ab has identical antigen specificity to the original mIg on the B cell.
•Differentiation to the plasma cell occurs about 4 days after immunization.
•Initially IgM is secreted.
•Later in the response naïve B cells undergo isotype switching.
Isotype Switching (switch recombination)
•The process by which B cells expressing IgD and IgM become modified at the genomic level.
•They start to produce other isotypes (IgA,IgE or IgG)
•Specificity is maintained as only the H chain constant region is switched.
•Specific T cell derived cytokines present in the microenvironment influences isotype switching.
•Class switching is irreversible.
-The segment of DNA between the 'old' H chain C region and the 'new' H chain C region is deleted
- Occurs at one week
Affinity Maturation (somatic mutation)
•With time following immunization there is a gradual accumulation of higher affinity antibodies.
•Due to random somatic mutation occuring in the rearranged VDJ gene regions of heavy chains and VJ regions of light chains -- point mutations
•Somatic mutation occurs in germinal centres about 7-10 days following Ag exposure.
•Any autoreactive clones which are generated by this mutation are deleted.
Generation of memory B cells
•Differentiation of memory cells from activated B cells takes place in the germinal centres.
•Starts about 7 days after stimulation.
•This is the time during which affinity maturation and isotype switching is occuring.
--> Memory B cells usually have higher affinity receptors and different isotype (other than IgM) from the originally stimulated naïve B cell. --> allows for a stronger response of second exposure with a higher Ab titer
•Some memory B cells remain in the secondary lymphoid tissue,
-most recirculate between blood, lymph and sites of previous antigen encounter --> secondary immune response can occur anywhere in body
- Entry into lymph nodes occurs via afferent lymphatics rather than HEV because they have less L-selectin
- Survive for weeks or months without further stimulation and are not in the cell cycle
-- Survival due to either stimulation by follicular dendritic cells that have trapped Ag/Ab complex OR sIgs on other B cells (idiotype) is recognized as Ag (called the network theory of regulation of the immune system)
Optimal activation of B cells
Requires co-stimulatory signals from ligation of CD19 (pan B cell marker)
•CD19 comprises part of the B cell co-receptor complex with CD21 and CD81.
•Also requires stimulation of CD40 by CD40L (CD154) present on activated T cells.
T-Independent Antigens
•Induce B cell activation in absence of T cell help.
•Two classes of TI antigens
-Type I e.g. bacterial cell wall lipopolysaccharide (LPS)
•Low doses acts as antigen
•High doses is mitogenic.
-Type IIe.g. pneumococcal polysaccharide (PP) from Streptococcus peumoniae.
•TI antigens do not stimulate memory cell formation and generally only cause IgM production
Low CD4+ cells, HIV infected. What induces a normal immune response?
Think T cell independent Ags
B Cell Regulation
•Important to have some regulation on Ab production, to avoid excessive generation of Ig.
•B cells possess FcRIIB (CD32) which binds antigen bound IgG.
•The result of interaction of IgG with the receptor is inhibition of antibody formation.
•A negative feedback inhibition.
Polyclonal activators - proliferation of many clones of lymphocytes
B cells: Pokeweed mitogen, high concentration of LPS
T cells: concanavalin A (con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM)
activate a subset of T cells
Bacterial products that are non-Ag specific, but non-polyclonal
• trigger oligoclonal activation.
• Between 2 -20% of clones are activated.
• bind to the Vβ region of the TCR. ***
•There is no processing of the superantigen by the APC.
• Eg. staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1)
TCR structure and diversity
The Variable region of the polypeptide Va is encoded by V, and J gene segments (similar to the k or l chain of BCR)
•The Variable region of the polypeptide Vb is encoded by V, D and J gene segments (like the variable region of Heavy chains)
•The process of somatic recombination occurs in developing Thymocytes within the thymus.

•Diversity of TCR occurs due to:-
-Inheritance of multiple germline genes
-Combinatorial association (Somatic Recombination)
-Junctional Diversity (generated by addition or deletion of bases during the recombination process)
-Random selection of the and chains to be combined in the TCR
•Total diversity of TCR is estimated at ~ 10^15
T cell development
1. Start off with a CD2 cell
-the somatic recombination of the b chain and allelic exclusion
expression of pre-TCR complex along with CD3
-CD2 antigen expressed (also on NK cells)
--> CD2, CD3, and pre-TCR

2. expression of CD4 and CD8 on same thymocyte, 'double positive' thymocytes
-->CD2, CD3, pre-TCR, CD4, CD8

3. - Somatic recombination of a chain
-Expression of the TCR complex
-Selection process ~ positive & negative selection
--> CD2, CD3, CD4, CD8, TCR

4. Lineage determination occurs
-Cells become committed to expression of either CD4 or CD8
-Single positive thymocytes.
-The remaining cells enter the medulla

5. Within the medulla, the negative selection process continues - the remaining thymocytes are exposed to bone marrow derived dendritic cells and macrophages, rich in Class I and II MHC
-CD4 or CD8 interaction with MHC molecules also occurs.
-The few cells remaining are mature naïve CD4+ or CD8+ T cells. Now free to enter the periphery.
-Referred to as Thp and pCTL cells - CD4+ nTreg, CD4+ Thp, CD8+ pCTL
Positive and Negative Selection of T cells
Results in central tolerance
- In the cortex
- Selects T cells that recognize self-MHC - avidity

Death by neglect:
- Not enough avidity/lack of recognition of MHC

- Starts in cortex, continues in medulla
- Too much avidity of T cells for MHC on thymic epithelial cells (cortex) or macrophages from bone marrow (medulla - more finely tuned)
- potentially auto-reactive T cells -- are killed
•Mutations in the AIRE gene reduce or eliminate the function of the AIRE protein -- implicated in autoimmunity (especially of endocrine origin - APECED syndrome)
•In the thymus, this protein causes ectopic expression of proteins normally restricted to specific tissues e.g. insulin. (so T cells are exposed to proteins and MHCs of TEC)
•Peptide fragments of these proteins are presented on the thymic epithelial cells in the peptide binding cleft of self MHC molecules.
•Developing T cells with TCRs recognising these with high avidity are negatively selected.
•This reduces the likelihood of developing autoimmunity.
nTREGs (natural T regulatory cells)
- CD4+,CD25+ (the chain of the IL-2R) and FOXP3+ (a nuclear transcription factor).
•They exit to the periphery and function as regulators of autoreactive T cells that have evaded the negative selection process in the thymus (peripheral tolerance to self Ags)
•nTregs also negatively control immune responses to 'non-self' antigens.
•Reduced numbers of nTregs or reduced expression of FOXP3 predisposes to autoimmune disorders and inflammatory diseases such as asthma and colitis
•Depletion of nTregs enhances immune responses to pathogens (eg. tumors).
• suppress:-
-A) transcription of a number of genes including IL-2 gene.
-B) proliferation of effector CD4+ and CD8+ cells.
-C) antibody production by B-cells.
-D) B-cell proliferation.

Two models of suppression seem to occur:
A) Bystander Suppression -the activated nTreg cell is able to suppress other effector cells with TCRs recognising different antigen specificities (inhibiting neighboring T cells)
B) Infectious Tolerance -modulation of the APC activity following interaction with a Treg, which recognises a particular Class II MHC:peptidecomplex.
•Other naïve CD4+ T cells interacting with different Class II MHC:peptidecomplexes on the same APC are suppressed.
•All of the epitopes are part of the same antigen and are displayed on the same APC surface.
Naive T cell trafficking to lymph nodes
- under the influence of adhesion molecules.
-The endothelial cells of HEV glycocalyx on their lumenal surface that serves to trap molecules.
-HEV possess addressins on their surface, which can act as 'homing receptors' for lymphocytes

Extravasation of naïve lymphocytes at sites of HEV requires:
-Interaction of L selectin on lymphocytes with ligand on endothelial cells (GlyCAM-1) -- rolling initatiation
-Activation of integrins (e.g. LFA-1) on the lymphocyte surface, producing firm adhesion to endothelial surface.
-Lymphocyte secretion of matrix metalloproteinases which degrade the subendothelial collagen to allow transendothelial migration into lymph nodes
•Should the naïve lymphocyte not encounter antigen in that lymph node it leaves via the efferent lymphatics and continues 'immunosurveillance' elsewhere.
•One cycle of the lymphocyte around the body takes between 1-2 days.
Thp Activation
•Thp cells encounter the foreign antigen presented to them by APC ~ generally Dendritic Cell in 1 response
•Th cells are Class II MHC restricted ** -- exogenous peptides
•The interaction of costimulatory molecules and their ligands is a requirement for activation of the Thp cell -- enhance the avidity of the peptide/MHC interaction with the TCR.
•In the presence of these interactions:
-the Thp cell starts to express IL-2R.
-the cell also secretes the cytokine IL-2
-interaction of IL-2 with IL-2R induces clonal expansion of the antigen stimulated T cell -- Th0 cells differentiate
-Interaction of CD28 with B7 (CD80/86) is crucial as the m-RNA for IL-2 is stabilised as a result. **
Th0 cells to Th1 and Th2 cells
- IL-2 driven clonal expansion of the Thp cell leads to Th0 intermediate.
•Th0 cells secrete cytokines common to both subsets (IL-4, IL-2, and IFNγ)
•IL-12 stimulates NK cells to secrete IFNγ
•Mast cells secrete IL-4 (trigger mechanism unclear)
•If IFNγ concentration is high then Th1 cell predominates.
•If IL-4 concentration is high then Th2 cell predominates.
•Th1 cells secrete Type 1 cytokines - stimulates macrophages, CTL, and NK cells
--immunity for viruses, fungi, parasites, and intracellular bacteria, also hypersensitivity
--IgG2A ispotype switching
--IL-2, IFNγ and TNF
•Th2 cells secrete Type 2 cytokines - stimulates B cells and plasma cells
--immunity for extracellular bacteria and parasites
--isotype switching to IgG1 and IgE (IL-4)
--IL-4, IL-10, IL-13, TGFβ.
Adaptive-induced Tregs (a/i Tregs)
- a/i Tregs arise from activated Th0 (nTregs from thymus) in presence of TGFb
• a/I Tregs are CD4+ CD25+ and FOXP3+ (Th0 only CD+)
• control immune responses to both foreign and to self antigens by down regulating effector CD4+ and CD8+ T cells
T Regulatory Type 1 cells (Tr1)
• secrete large concentrations of IL-10, and having a cytokine profile similar to Th2 cells.
• both IL-10 and IFNg play a role in their differentiation.
• peripheral tolerance.
•Tr1 cells are abundant in the intestine - maybe role in induction of tolerance to dietary antigens
Th3 cells (Tr2 cells)
•A subset of CD4+ regulatory T cells which primarily secrete TGFβ.
•Oral tolerance is established in the MALT - the lamina propria.
•suppress both Th1 and Th2 effector cells.
•TGFβ also modulates B cell function, by increasing the production of IgA.
- not sure what cytokines induce their differentiation
Th17 cells
•These are effector T cells initially shown to arise from Th0 cells in the presence of TGFβ PLUS IL-6 or IL- 21.
• three distinct stages in their generation:
-1. Differentiation (involves TGFβ+ IL-6/TGFβ+ IL-21)
-2. Amplification (involves IL-21 autocrine amplification)
-3. Stabilization (involves IL-23 from activated APCs)
• immune responses against extracellular pathogens that are not eliminated by Th1 or Th2 cells - imp. in fungal infections
• secrete the proinflammatory cytokines IL-17A, IL-17F, IL-21 and IL-22.
• induce the secretion of inflammatory mediators by macrophages and other inflammatory cells -including cytokines, chemokines and MMPs.
•Autoreactive CD4+ Th17 cells are highly pathogenic, causing inflammation and severe autoimmunity in animal models.
•Down regulation of Th17 cells is proposed to be mediated by the type 1 cytokine IFNg and the type 2 cytokine IL-4.
T cell regulation
•Role of B7 (CD80 and CD86).
-CD28 on the T cell interacts with B7 on the APC - co-stimulatory signal.
-The interaction is required for stabilization of mRNA for IL-2.
•2-4 days after activation the T cell starts to express a new surface molecule CD152.
-This has a higher affinity for B7 than does CD28 ~ it induces down regulation.

- IL-4 from Th2 inhibits Th1
- INFg from Th1 inhibits Th2
- IL-10 from Th2 cells inhibits the secretion of IL-12 from APC -- blocks action of IL-12 on NK Cell, preventing the secretion of IFNγ.
• membrane bound protein present on T cells, B cells and Dendritic cells.
• CD200R is expressed mainly on myeloid cells (monocytes, macrophages and dendritic cells)
•This receptor is also expressed on some T cells (more on CD4+ and memory cells)
-CD200R expression is up-regulated on both CD4+ and CD8+ T cells after stimulation.
•No CD200 - autoimmunity, too much CD200 - tumor
•Anti CD200 would block the interaction of CD200 with CD200R
CD8+ pCTL Activation
•pCTL forms a conjugate with presented endogenous peptides on class I MHC molecules.
•Class I molecules present on any nucleated cell (target cell).
•Numerous molecular interactions occur including TCR/MHC I peptide complex, CD8/MHC I (a3 domain).
- conjugate formation stabilized by high avidity interaction of adhesion molecules CD2 and LFA-3, LFA-1 and ICAM (1-3)
- optimal activation requires CD8 to bind to class I MHC at a site different from TCR binding
• following conjugate formation - expression of IL-2 R.
•This can interact with the IL-2 derived from Th1 cells recognising peptides on APCs.
•Major stimulus to the generation of CTL from pCTL is action of IL-2 and IFNγ from Th1.
•The progression from pCTL to CTL occurs over ~ a week, following detachment from the presenting complex.
•There is clonal expansion of pCTLs which express IL-2R (paracrine action).
•Mature CTL are capable of killing autologous infected cells, bearing bearing viral peptides which stimulated the original cell.
CTL killing
•Involves recognition of suitable target cells and delivery of the 'lethal hit'
•Role for Perforin and Granzyme B in activating the apoptotic cell death of targets.
•Having delivered the death message the CTL can move off and kill further infected autologous cells.
Viral evasion methods
Particularly evade CTL
-Herpes simplex virus: produces a protein which binds to cytosolic portion of TAP.

-Epstein Barr virus: inhibits activity of the cytoplasmic proteosome. also produces a viral protein which is a structural homolog of IL-10. IL-10 is a Th2 cytokine which inhibits the Th1 cell (via inhibition of IL-12 secretion from activated macrophages & dendritic cells).
The Th1 cell is the appropriate cell to provide help (cytokines IL-2 and IFNg) to cause differentiation of pCTL to CTL

-Cytomegalovirus: encodes proteins that redirect newly synthesised Class I MHC molecules from ER into the cytoplasm, where they are degraded by the proteosome.
Determinant selection/non-responsiveness
• MHC molecules present on the cell do not bind a particular peptide.
•If the peptide can't be presented to T cells no immune response can be generated against it.
Damage associated molecular patterns e.g. uric acid crystals, cholesterol crystals.
NALP3 (NLR) and Inflammasomes
Becomes active when it binds an appropriate ligand (PAMP or a DAMP).
•On activation the assembly of the NALP3 inflammasome is initiated.
•This is a large intracellular complex comprising the activated NALP3 and the pro-Caspase1 (an inactive form or zymogen)
•As a result the pro-Caspase-1 is activated (Caspase-1 or IL-1 Converting enzyme) and this active enzyme cleaves pro-IL1, pro-IL18 and pro-IL33 into their active forms.
•Hence the inflammasome plays a crucial role in innate immunity.
•IL-1 is a key mediator of inflammation.
•IL-18 along with IL-12 stimulates NK cells to secrete -IFNg.
•IL-33 plays a role in allergic inflammation and asthma. Immune cell source of IL-33 not clear
Vascular Endothelium
•The normal endothelium is an inert surface.
•cytokines released by macrophages (IL-1 and TNF) after exposure to pathogens, activate the endothelium.
•Role of IL-1 & TNF in inflammation:
-Expression of E Selectin on endothelium (to allow rolling)
-Expression of VCAM-1 and ICAM-1 on endothelium.
-Induce secretion of IL-8 (CXCL8 - chemotactic for neutrophils) and MCP-1 (CCL2 - chemotactic for macrophages/monocytes) by endothelial cells.
-Autocrine and paracrine activation of Macrophages
•Role of TNF
-Enhanced cytotoxicity of Macrophages (induces iNOS)

TNF secreted from Th1 activates endothelial cells and macrophages to secrete TNF and IFNg (iNOS and NADPH oxidase) - increase Macrophage toxicity
•During continued inflammation lymphocyte rolling, adhesion and diapedesis of T cells occurs at sites of inflammation (as for the earlier responding neutrophils)
•Activation of T cells increases their expression of integrins LPAM and VLA-4.
•These molecules interact with VCAM-1, producing firm adhesion.
resists both direct and opsonin mediated phagocytosis.
This organism is still susceptible to C' mediated lysis by generation of the MAC.
Individuals with MAC component deficiencies (e.g. C8 deficiency) are susceptible to infection with these encapsulated bacteria.
Shigella dysenteriae
produces a toxin which is associated with damage to the colonic vasculature, triggering bloody diarrhoea.
The organism binds to M cells and is endocytosed.
The organism can lyse the endosomal membrane and gain access to the cytosol, where they can replicate.
It spreads laterally from cell to cell in the epithelium.
Organisms which do not infect neighboring cells pass through the M cell into lamina propria.
Here they are phagocytosed by macrophages, which are induced to undergo apoptosis.
The organism hence causes destruction of the intestinal surface and the phagocytes
Borrelia burgdorferi
Causative organism of Lyme disease.
A number of organisms undertake antigenic variation in order to evade the protective immune response.
The external appearance of the organism varies subtly.
This means infections may persist for long periods despite mounting an immune response - due to alteration in cell surface lipoproteins.
The organism keeps one step in advance of the immune response.
Mycobacterium tuberculosis
an intracellular bacterium.
Some components of the complex cell wall prevent the fusion of lysosomes with phagosomes.
The organism can persist in the phagocytes e.g. alveolar macrophages.
Rickettsia rickettsii
causative organism of Rocky Mountain Spotted Fever.
Transmitted via dogs, rodents, foxes carrying ticks.
Tick attaches to the skin and begins blood meal.
Rickettsia attaches to receptors on endothelial cells and enters the cell in an endosome.
The organism can escape the endosome and enters the cytosol, spreading to adjacent cells or the extracellular environment.
The organism can be phagocytosed but is able to escape into the cytosol of the phagocyte.
Human Immunodeficiency Virus (HIV)
causative agent of AIDS.
Causes direct and indirect destruction of the CD4+ Th cells.
A number of receptors on cells can interact with HIV and allow entry into the cell:
CD4, CCR5 (Macrophages) and CXCR4 (Th)
Roles of Igs in immunodiagnostics
•IgG ~ often employed as reagent and assayed in vitro.
•IgM ~ often employed as reagent (an excellent agglutinator due to valency of 10), also often assayed in vitro.
•IgA ~ rarely employed as a reagent in testing.
•IgE ~ Rarely employed in testing. Allergen specific IgE may be assayed by RAST in allergy diagnosis.
•IgD ~ Almost never employed or assayed (levels may be monitored in cases of IgD myeloma)
- Ab must be bivalent
- Ag must be bi- or poly- valent
- Precipitate visualized in semi-solid agarose gel

- Precipitin curve:
- in general, as Ag is added, the amount of precipitate increases, until a certain point, and then declines
•Prozone: Area of antibody excess
•Zone of Equivalence: Both reactants (antigen and antibody) are in equal concentration
•Postzone: Area of antigen excess
Test for syphilis (venereal disease research lab test)
•Detects antibody to cardiolipin.
•Flocculation = a specific type of precipitation that occurs over a narrow range of [Ag]. The antigen consists of very fine particles.
•Serum or CSF
•assessed microscopically x100
- non-specific for anti-cardiolipin (could also be SLE, etc.) - need further testing
Oudin's Single Immunodiffusion
Gel-based immunodiffusion
-A monospecific antiserum is mixed with molten agarose and poured into a series of tubes and allowed to set.
-contains a uniform distribution of antibody molecules
-Liquid samples for analysis are pippetted onto the top of the gel and allowed to diffuse into it.
-Appearance of discs of precipitation in the gel indicate the presence of the antigen
- Lines of precipitation appear at 'zone of equivalence' in each tube - Position/ranking is dependent on amount of specific antigen in each test sample.
- qualitative and could be semi-quantitative.
- Polyspecific antisera to indicate presence of > 1 antigen in sample - No means of indicating which 'zone of equivalence' was due to each Ab and Ag combination.
Mancini's Single Radial Immunodiffusion
•Dependent on radial diffusion of soluble antigens into semi-solid gels containing monospecific antibody.
•Allows quantitation of antigen level with reference sample of the antigen (standard curve).
•when the diffusion of the antigen is complete, the diameter squared of the precipitate is linearly proportional to the [Ag] in the well.
•Standard curve generation: A range of dilutions of the reference are made and d^2 is measured at completion (speed of diffusion depends on the size of the molecule)
- Once the standard curve is constructed, the concentrations of antigen in the 'test' samples is calculated by measuring d^2 and dropping a perpendicular onto the [Ag] axes.
- Multiplication of the [Ag] by the dilution factor provides the final [Ag]
Ouchterlony Technique
•Double Immunodiffusion - both antibody and antigen diffuse towards each other in the gel.
-As equivalence is reached a visible line of precipitation (a precipitin line) forms.
•Pattern of identity: two antigens share identical epitopes. The two precipitin lines produce a single curved line of identity.
•Pattern of nonidentity: the antigens are unrelated. The antiserum forms separate precipitin lines with each antigen, which cross
•Pattern of partial identity: two antigens share epitopes but one possesses unique epitopes. The antiserum forms a line of identity with the shared epitopes and a curved spur with the unique epitope(s). The spur points towards the antigen which is partially recognised by the antiserum.
•speed of movement of an antigen into an antibody gel increased by applying an electromotive force.
-Advantage of speed BUT needs specialised equipment

Rocket Immunoelectrophoresis (Laurell)
•As antigen is driven into the gel, lateral precipitation occurs with antibody in the gel.
•Rocket shaped precipitates are formed.
•The height of the rocket is linearly proportional to the [Ag] concentration in the sample applied.
•Accurately diluted reference samples employed to produce the Std curve.
Antigen concentration from graph x dilution factor = True [Ag]
•A two stage process which is usefully employed in the diagnosis of myeloma or humoral immunodeficiency states.
•In myeloma IEP indicates which isotype is forming the paraprotein seen on serum electrophoresis
•IEP is a combination of 1. electrophoretic separation of proteins in serum and 2. immunoprecipitation of proteins with either polyspecific or monospecific antisera
- 1. Initial stage is serum electrophoresis of normal or control serum and patient's serum.
2. Rabbit anti -human serum is added and over a period of a few days precipitin lines form.
Direct agglutination
Occurs when Ag is found naturally on the surface of a particle e.g on bacterial surface or on rbc as in ABO grouping
If you put anti-A antibody into blood, you will see agglutination in type A and type AB blood
Passive agglutination
Particles are coated with Ag not normally found on their surface e.g tanned red cells with Thyroglobulin.
Then these RBCs are placed in blood, if antibody exists, there is agglutination
Reverse passive agglutination
Antibody rather than antigen is attached to the surface of the particle usually latex beads ~ often used for the detection of specific microbial antigens
If these Ab coated particles are placed in blood with microbial antigens, agglutination will occur
Agglutination inhibition
Based on competition between particulate and soluble antigens for a limited number of antigen binding sites.
•A lack of agglutination indicates a positive test. The classical example being the pregnancy test based on hCG. Latex particles coated in hCG.

1. Incubate a patient sample with antibody (eg. anti-hCG).
2. If patient has antigen (hCG), these will bind
3. Add antigen coated latex particles (hCG)
4. If patient had the hCG, then there will be no binding and no agglutination because all the spots are taken up. -- Positive pregnancy test = no agglutination
5. If patient does not have the hCG, there will be agglutination with the anti-hCG latex particles. -- Negative pregnancy test = agglutination
Bacteria are used as the inert particles to which Ab is attached. S.aureus often employed due to possession of protein-A which binds Fc portions of IgG.
1. Put specific antibody with bacteria, which will take it up and show Fab region
2. Put in patient's blood
3. If there is antigen present, agglutination will occur
Coombs Test - Direct
Developed to overcome the zeta potential and allow the detection of IgG antibodies on red cells which themselves are unable to agglutinate the cells (incomplete).
-Rabbit anti-human globulin (Antiglobulin) is used

-Direct Coombs' Test (Direct /antiglobulin Test) ~ detects antibody which are already bound (in vivo) to the surface of the red cell.
•E.g. autoimmune haemolytic anaemia (IgG causing premature destruction of the individuals own red cells)
•HDN (Erythroblastosis foetalis) newborn's red cells have maternal anti-D on them.

- Just add anti-human globin (IgG), and if there is antibody present on the RBC (for example, anti-A), then there will be agglutination
Indirect Coombs Test (Indirect Anti-globulin Test)
Employed to detect:
•The presence of a particular antibody in a patient's serum
•Or to type patients red cells for specific blood group antigens.

1. Reagent red cells and patient's serum antibody are incubated at 37°C and then washed with saline to remove any uncombined Ab
2. Anti-human globulin is then added to cause visible agglutination if Ab has bound onto the cells in the first step.

•The IAT is an important aspect of the 'cross-match'.
1. Recipient's serum is incubated with donor red cells.
2. If pre-existing IgG antibody to a red cell antigen is present they will attach to the donor cells.
3. After washing in saline, AHG is added.
-Agglutination indicates presence of atypical Ab in the potential recipient.
-Absence of agglutination indicates compatability of donor and recipient.
-**Coombs' positive cells are then added to check that the Coombs' reagent is functioning correctly (Positive control)
•If an atypical antibody is detected in the cross match the specificty of the antibody needs to be identified.
•Patient's serum is incubated with a panel of red blood cells of known antigen profiles.
•Indirect Coombs' Testing after incubation and washing should allow the identification of the specificity
Haemolytic titration (CH50) assay
- functional assay of complement activity.
- measures amount of patient serum required to lyse 50% of a standardized concentration of antibody sensitized srbc.
-Absence of any component or components will result in an abnormal CH50 result, essentially reducing the result to zero.
- Titer is expressed in CH50 units, which is the reciprocal of the serum dilution which was able to lyse 50% of the sensitized cells.
- 0-100 CH50 units: Absent or low activity
100-300: Normal
> 300: High activity
• It does not identify which component(s) are deficient.
•Abnormality needs to be further investigated by individual C' component assays.
•Assay systems employed for individual components include RID, Rocket IEP, latex agglutination assays or ELISA.
Complement Fixation Tests
•Complement is utilised as a reagent for the detection of specific antibody or antigen in a sample.
• Detection of viral and fungal antibodies.
1. Patient's serum is heated at 56°C for 30 minutes.
2. Dilutions of the serum are combined with known antigen and a measured amount of guinea pig complement.
3. If specific antibody is present in the sample it will combine with Ag to form immune complexes.
4. The complement will be 'fixed' (C1q binding to Fc fractions of the Ig)
5. Indicator step. Addition of the sensitized cells.
6. If the guinea pig complement has been fixed in the first step there will be none available to lyse the cells.
7. Failure to lyse cells indicates a positive test!
•Results for CFTs are expressed as the highest dilution producing no lysis of the cells.
•The CFT can be performed in microtitre plates as can haemagglutination techniques.
ELISA Techniques
•These techniques are similar in principle to Radioimmunoassay (RIA) but they have a number of advantages:
-No radioisotope is needed Safer
-No expensive counting equipment ~ employs spectrophotometer or automated plate reader less costly.
-Similar sensitivity to RIA.
•Antibodies are conjugated with an enzyme rather than a radio isotope.
•Antibodies commonly employed include alkaline phosphatase, horseradish peroxidase and β-Galactosidase.
Indirect ELISA
1.Antigen coated plate
2.Wash to remove any free antigen
3.Add Std Ref dilution to wells and diluted samples (usually in duplicate) - Serum or other sample suspected of containing Ab to the Ag is added and allowed to react.
4.Wash to remove any unreacted Ab
5.Add 2°antibody (enzyme conjugated anti-Ig)
6.Wash to remove excess/unreacted 2°antibody
7.Add substrate for each well and measure colour development (absorbance @ nm) with spectrophometry - Intensity of colour developed reflects amount of enzyme in the well
8.Plot Std curve, estimate unknowns

•Indirect ELISA can be employed to detect Ab to HIV.
•Individuals infected with HIV produce antibodies to envelope and core proteins of the virus.
•Usually serum Abs can be detected by this technique within 6 weeks of infection.
Sandwich ELISA
1. Antibody coated plate - add antigen which is captured by the antibody
2. Wash to remove any unbound antibody
3. Add Std Ref dilution to wells and diluted samples (usually in duplicate) - antigen
4. Wash to remove any unreacted Ag
5. Add 2°antibody (enzyme conjugated antibody directed against second epitope)
6. Wash to remove excess/unreacted 2°antibody)7. Add substrate and measure colour development (absorbance @ nm)
8. Plot Std curve, estimate unknowns
Western Blotting
Detects specific proteins in cell lysates - used to detect antibodies to specific proteins
•Cells are placed in detergents and the proteins solubilized.
•Lysate is subjected to SDS-PAGE to separate the proteins according to MW.
•The separated proteins are then 'blotted' onto nitrocellulose membranes.
•Antibodies to the specific antigens are added to the membrane and allowed to interact.
•Presence of Ig and hence protein is detected by addition of enzyme conjugated anti-Ig
Immunofluorescence Microscopy
•A useful aid in the diagnosis of autoimmune disorders.
•Usually involves an indirect IF method, employing a second Ab to detect the presence of an autoantibody in a patient's blood.
•Incubate a dilution (usually 1/10) of serum on a microscope slide, containing thin sections of a composite block of tissue.
•The block of tissue may consist of human thyroid tissue, along with murine kidney, stomach and liver.
•During incubation any reactive antibodies in the sample will bind to specific sites in the block.
•After suitable washing step to remove any unbound antibodies, the 'tagged' fluorescent anti Ig is added.
•The fluorescent Ab is generally a rabbit anti-human Ig tagged with either:
-Fluorescein Isothiocyanate (FITC)
•After appropriate washing to remove any unbound second Ab, the slides are examined in UV microscope.
Macrophage chemotaxis process
1. Circulation
2. Leave vascular system
3. Migrate to areas where needed (injury/infection)
4. Margination (postcapillary venules)
5. Diapedesis (transmigration)
6. Chemotaxis (chemokines)
Which cytokines are involved in inflammation? And what do they do to the vascular endothelium
IL-2 - macrophages
TNF - macrophages, Th1

1. Expression of E-selectin (cell rolling) on endothelium
2. Expression of VCAM-1 and ICAM-1 on endothelium and enhanced ICAM-2
3. Induce secretion of IL-8 (CXCL-8) and MCP-1 (CCL2) by endothelial cells
4. Autocrine and paracrine activation of macrophages leading to secretion of cytokines (IL-1, TNF, IL-6, IL-12)
5. TNF also enhances cytotoxicity of macrophages
p75 Fusion Protein
Binds TNF before it could bind to macrophages
Side effects:
- lymphomas
- fungal infections
CTLA-4/CD152-Ig (fusion protein)
Treats Rheumatoid arthritis
- Inhibits the co-stimulation of T cells by cell cycle arrest
- Targeting T cells because they secrete TNF which causes the inflammation
- Binds to APC via B7 (CD80/86) and prevents T cells (CD28) from binding, thus T cells not activated (IL-2 mRNA is not activated)
- In addition, after the Ig binding to the APC, phagocytes would engulf them, depleting B7 cells
- Side effects: autoimmunity; exacerbation by blocking endogenous CTLA-4 activity and blocking Treg cells (CD4+ CD25+)
Multiple Sclerosis
-Autoimmune disease which targets myelin of nerve tissue
-Delayed type hypersensitivity (t-cell mediated)
-Lesions occur when activated T cells (and other inflammatory cells) CROSS THE BBB
Molecular mimcry
A potentially autoreactive T cell, possessing TCRs that recognize both a foreign (viral) peptide and a self-peptide, is activated by a virus-derived peptide. Thus, in addition to mediating an antiviral response, the T cell is also capable of mediating self-directed responses.
Anti-VLA 4
Treatment for MS
- VLA-4 made up of CD49D (a chain) and CD29 (b1 chain)
- Anti-VLA 4 specifically targets the a chain
- Prevents firm adhesion so there is no crossage of the BBB
Anti-IL2R (anti-CD25)
Treatment of MS
- Macrophages are key mediators in MS - secrete TNF, IL-2, INFg
- IL-2 is a growth factor for T cells
- IL-2R is expressed on T cells after presentation by DCs
- Prevents activation of more T cells
- Side effect: Tregs are targeted (which helps in downregulation of immune response)
Anti-IL 12 (Anti-p40)
Treatment for MS
- IL-12 is normally released by DCs to activate NK cells to release INFg to grow more Th1 cells (TNF, IL-2, INFg)
- IL-12 has two parts: p40 and p35. Anti-IL 12 targets p40
- Prevents formation of Th1 cells
- Skin disease causing itchy and sore patches, thick red skin, silvery patches
- Skin cells rise too fast - high turnover.
Anti-p40 (anti-IL12/IL23)
Treatment for psoriasis
- p40 on both these cytokines
- Prevents formation of Th1 (IL-12)
- Prevents Th17 stablization (IL-23)
- Th17 produces IL-17 which induces proinflammatory enzymes and is a chemotactic for neutrophils
Systemic Lupus Erythematosus
-Immune complex mediated
- Facial rash, sensitivity to sunlight, cold/numb, white/blue/red, joint pains, discoid rach, periungual erythema, oral ulcers, lupus vasculitis, lupus nephritis
- Soluble antigens - Antinuclear antibody (ANA), Anti-dsDNA, antiphospholipid antibodies (lupus anticoagulant) - in vivo actually increases coagulability, RNA (anti-Sm)
- Autoantibodies, tissue damage due to immune complex deposition and associated immune responses
- Pathology secondary to complement activation - Immune complexes are associated with complement activation (C3b is an opsonin and C5a a chemotactic) and attempted IgG opsonin mediated phagocytosis
- Frustrated phagocytosis bc neutrophils can't phagocytoze the IgG
Treatment for non-Hodgkins lymphoma and SLE (off-label use)
- CD20 is a B-cell restricted cell surface Ag (from pre-B cell stage on, not on plasma cell or pro-B cell) without a clear function or ligand; maybe TK/Ca channel; not shed or internalized (not in circulation)
- For SLE, need only 3 doses for remission
- After Anti-CD20 (Fcg) binds, phagocytic opsonin-mediated phagocytosis, ADCC (NK cells), complement activation (phagocytosis and MAC)
Anti-CD200 Antibody
Cancer treatment - chronic lymphocytic leukemia
- CD200 expressed on T cells, C cells, and DCs
- CD200 expression on tumor cells suppresses anti-tumor cytotoxic immune responses
- CD200R expressed on macrophages and DCs, and some on T cells (4>8 and M>E)
- Prevents suppression of immune response on tumor cells
Pulsed dendritic cells
Cancer therapy
- Tumor lysate, purified tumor Ag, or tumor gene transfer placed in presence of Dendritic cells so that DCs will pick them up to present to T cells
- Injection of DCs along with tumor lysate, etc. back into patient
- cross presentation needed to activate CTLs (bc DCs normally present on MHC II to Th cells). In order to kill tumor, we need CTL cells (MHC I recognition)
Cancer therapy
- DCs from patient are cultured to increase number
- Damage tissue (heat, radiation, cryotherapy) to induce cell death and release tumor antigens
- DCs are injected into the damaged tissue (tumor) to pick up the antigens and activate immune response to that specific tumor
Gene Engineering/irradiated tumor cells and GM-CSF gene
Cancer therapy
1. Remove tumor cells and irradiate in vitro so they can't divide
2. Insert gene that encodes GM-CSF (or IL-2, etc.) into the irradiated tumor cells
3. Inject modified cells into the patient - GM-CSF will be secreted from these cells
4. DCs and phagocytes are attracted to site
5. Endocytosis of irradiated tumor cells
6. Migration of DCs to regional LN
7. Presentation to T cells (and B cells)
Regulatory T cells (Tregs) Depletion
Cancer therapy
- Tregs usually suppress immune response
- Ex-vivo cell sorting and depletion of Tregs
- Target Tregs to prevent suppression
- Side effect: autoimmune (ntregs), inflammation (a/i tregs)
Recombinant cytokine therapy
Cancer therapy
-IL-21 - amplification of Th 17 cells, which produces IL-17A and IL-17F
--These cytokines induce proinflammatory cytokines, induce chemokines (e.g. IL-8), induce metalloproteinases, and IL-17A is a chemotactic for neutrophils
-IL-2 - T cell clonal expansion, converting pCTLs to CTLs, and enhancing NK cell cytotoxicity (LAKs)
--IL-2 receptors on CD 25 cells
--however, IL-2 causes toxicity by vascular permeability, causing death
Adoptive Cell Transfer
- Melanoma
- Ex vivo expanded infiltrating lymphocytes (TILS)
- Lymphodepletion
- Adoptive transfer peptide specific T cells, vaccine, and IL-2
- Side effects: vitiligo (killing melanocytes) and autoimmune destruction of melanocytes in the eye (severe ocular autoimmunity)
How does the immune system destroy tumors?
1. Opsonin-mediated phagocytosis
2. NK cells - ADCC and KAR
3. CTLs - needs IL-2 for pCTL to CTL

Other cells that play a role:
1. DCs (Ag presentation)
2. CD4+ T cells (cytokines)
3. B cells (Abs)
Hepatitis A
- RNA vaccine
- short (2 wks) incubation period
- transmitted through food and water
- symptoms: anorexia, fever, ab pain, jaundice
- no long term/chronic infection, not fatal
- no treatment
- IgM anti-HAV found in blood after exposure
- 1 dose of vaccine provides some protection, but V + Ig protect for 3 months
Hepatitis B
- DNA virus
- transmitted through sex, needles, blood transfusions, dental/cosmetic/ procedures (tattoos, piercings)
- given in series of doses over 6 months
- some protections after 1 or 2 doses
Recombinant vaccines
- Made by producing multiple copies of a single virus or bacterial protein with assistance of a vector (yeast or bacteria)
- safe for immunocompromised patients
- eg. hepatitis, pertussis
Typhoid Fever
- Salmonella enterica typhi
- fecal-oral transmission
- incubation short 1 week
- fever, malaise, diarrhear, death due to bleed/perforation of intestines
- treat with antibiotics for those infected
- oral vaccine (live attenuated) and polysaccharide vaccine given IM available
- only 50-80% effective
Passive Immunization
1. Natural: IgG crossing placenta; IgA and IgG in breast milk
2. Artificial: gamma globulins (immunoreactive serum) for pre-exposure prophylaxis or pre/post exposure prophylaxis (to prevent disease in immunocompromised)
Active Immunization
1. Unintentional: pathogen exposure to live virulent organism
2. Intentional: Pathogen exposure by
- killed microbes
- attenuated microbes
- live natural microbes
- modified toxins
- conjugated molecules
- recombinant proteins
- naked DNA
Substances added to vaccines to make them more immunogenic but don't themselves confer immunity
- inorganic salts (aluminum hydroxide)
- immune stimulating complexes (liposome)
- submicron oil-in water emulsion
- bacterial cell products (inactivated bordatella pertussis)
Killed/inactivated microbes vaccine
- polio (salk), hep A, pertussis
- paralysis and death
- infection via fecal oral route
- Salk Polio Vaccine - doesn't generate mucosal immunity since it is injected. Infected individuals can still spread disease, so not that effective
- Pertussis - toxins are potential problem
Live attenuated vaccines
- measles, mumps, rubella, polio (sabin), varicella, arbovirus (tellow fever), mycobacterium bovis (not that effective)
- Problem: reversion, vaccine induced polio
Toxoid immunization
- tetanus, diphtheria
- safe and effective even for immunocompromised
Conjugated molecule vaccine
- streptococcus pneumoniae, hemophilius influenzae (type b)
- carbohydrate plus protein

hemophlilus influenzae type b
- major cause of meningitis under 5
- surrounded by a polysaccharie capsule
- PRP within capsule is immunogenic

streptococcus pneumoniae
- major cause of pneumonia, middle ear infections, meningitis
- surrounded by polysaccharide capsule (gel)
- Abs exacerbate disease due to lysis of bacteria which leads to release of products that stimulate too vigorous inflammatory response, which leads to endotoxic shock
Immunosuppression and vaccines
- Live attenuated vaccines are contraindicated for patients with severe impaired cellular immunity
- If a patient is on a dose of equivalent of 2mg/kg or at least 20mg/d of prednisone for >2 weeks do not give live virus vaccines. Wait 2-4 weeks after a course of steroids to give vaccines
Hyperacute rejection
-Occurs min to hours post-transplant
- primarily Ab-mediated
- depends on pre-formed Abs that recognize donor Ag - xenoantigens, pre-immune recipient
Delayed hyperacute rejection
- few days post tranplantation
- associated with activation of endothelial cells
- thrombosis
- procoagulants (fgl-2)
Acute rejection
-weeks after transplantation
- T-cell mediated
-Type 1 cytokines
Chronic rejection
- months-years post-transplant
- insidious phase of rejection
- increased production of non-specific growth factors (FGF, endothelial growth factor)
- chronic proliferation of CT and mesenchymal cells
- associated with type 2 cytokines
- refractory to immunosupression therapy
Ischemic/reperfusion injury - graft rejection
Induces chemokine and cytokine secretion by graft (donor) cells that
1. activate vascular endothelial cells - adhesion molecules, secrete IL-8, MCP-1
2. enter circulation - cytokines and chemokines activate to high affinity molecules on circulating leukocytes and cause extravasation of T cells into graft
3. alter vascular permeability - allows the extravasation where MHC II alloreactive CD4 T cells in the graft are activated and differentiate primarily to Th1 cells - mature CTLs activated
Tumor recognition
1. Tumor specific antigens
2. Tumor associated antigens
3. Over-expression of normal self proteins
Tumor specific antigens
- Molecules or proteins which are located on the surface of tumor cells
- specific for cancer cells and thought to be expressed de novo
- not found on normal cells or in minimal amount
- target for cancer therapy (bullet)
- MAGE-1, MAGE-3 - melanoma, normal testicular protein
- Surface IgG - lymphoma, normal B cell receptor
- NY-ESO-1 - breast cancer, normal testicular cancer
- GAGE - melanoma
- BAGE, PRAME, Caspase-8 - squamous
Tumor associated Antigens
- Reactivation of embryonic genes not normally expressed in differentiated cells
- Ags located on normal as well as cancer cells
- more common than tumor specific Ag
- non-specific but can be used to diagnose certain cancers
- oncogenic Ags or viruses that transform
- Carcinoembryonic Ag (CEA) - oncofetal Ag, COLON, breast, stomach; useful to monitor recurrence; present in non-malignant tissues (smokers, COPD, cirrhosis)
- AFP - oncofetal Ag; liver and testicular, pancreatic, gastric, lung; normally synthesized by fetal liver, yolk sac, GI cells; also in cirrhosis (leading to cancer)
- b-hCG - gonadal germ cells tumors; adenocarcinomas of the ovary (hydatiform/molar), pancreas, stomach, hepatomas; used to monitor efficacy of therapy; produced by trophoblast epithelium of placenta
Overexpression of self proteins
- Changes density of peptide presentation, allowing regulation by T cells
- self proteins gives a minimal immune response
- HER2/neu - serves as co-receptor of tyrosine kinase associated growth factors; involved in signal transduction and cell growth and differentiation; breast (bad prognosis if seen), ovarian, endometrial cancers
CTLA4 blockade
-Prevents downregulation of T cells (cancer therapy)
Rheumatoid Arthritis
-Immune complex mediated autoimmune disease
- primarily affects small joints of the body causing synovitis, joint deformity/redness, fatigue, fever, morning stiffness, vasculitis, pleural effusion, anemia, epicleritis, kidney disease, increased risk of stroke/MI
- chronic, inflammatory disease with no cure, systemic disease affecting one or many organs
- Rheumatoid factors: IgM (or IgG/IgA) Anti-IgG (Fc) antibodies
- may not be elevated or present in individuals with RA
- IL-1 and TNFa are significantly elevated - target when other treatment doesn't work
- diagnosis with aid of serological tests - RF, CRP, ESR
Pernicious Anemia
- Antibody mediated autoimmune disease
- Abs to intrinsic factor or parietal cells
- Lack of B12 leads to megaloblastic anemia
Goodpasture's Syndrome
- Antibody mediated autoimmune disease
- Anti-glomerular basement membrane Abs
- HLA allele associated with HLA DR2
- slightly more in males, younger patients
Myasthenia Gravis
- Blocking Antibody autoimmune disease
- Abs block the receptor for ACh - inhibits transmission of nerve signals and cause muscle weakness leading to difficulty breathing, death
- AChRs are endocytosed
- ptosis
Graves's Disease
-Stimulating Antibody autoimmune disease
- Abs against TSH receptors - stimulate thyroid function, leading to hyperthyroidism
- exophthalmos, pretibial myxedema, goiter
Wegener's Granulomatosis and Churg Strauss Vasculitis
- Stimulating Abs
- Anti-cANCA and anti-pANCA Abs
- IgG and IgM Abs (immune complex formation, complement activation, phagocytosis)
- Churg - pulmonary hemorrhage and infiltrates
Hashimoto's Thyroiditis
- Cell-mediated Autoimmunity
- initially produces hyperthyroidism and then leads to hypothyroidism
- initial destruction occurs when T cells infiltrate the gland and release contents of thyroid cells leading to hyperthyroidism
- Abs are: Anti-thyroglobulin and anti-microsomal Abs
Multiple Sclerosis
- Cell-mediated Autoimmunity
- Target is myelin of nerve tissue
- oligoclonal Ig bands detected in CSF of patients with symptoms of MS
- delayed type hypersensitivity (t-cell mediated)
- unidentified bright plaques in brain
Ankylosing Spondylitis
- Cell-mediated Autoimmunity
- HLA allele - HLA B27
- lots more males, younger
- loss of lateral flexion of lumbar spine due to stiffening of sacroiliac and spinal facet joints
Primary Immunodeficiency Disorders
- Inherited, genetic defect of T, B cells, phagocytes, complement system
Secondary Immunodeficiency Disorders
- acquired, variable etiology: viruses, meds, alcohol, disease states, stress, aging
Severe Combined Immunodeficiency
- X-linked SCID (g-chain mutation CD132 - signal molecule for many cytokine receptors); Adenosine Deaminase Deficiency (ADA); T-B SCID (Rag1, Rag2 defects); JAK3 Deficiency
- No development after lymphoid precursor cell (no B or T cells)
- fungal infection (white plaques in mouth)

Treatment: gene therapy
Side effect: leukemia like symptoms - Overproliferation of one of the T cells
Bruton's Agammaglobulinemia
- Primary immunodeficiency
- Absent or low levels of Igs
- X-linked - males only affected
- Maturing of B cells is blocked (pro-B to pre-B) - No B cells
- Btk (Bruton's tyrosine kinase) defect
- variety of infections occur in early childhood
- mortalities largely due to bacterial encapsulated orgs and certain viral infections
- live vaccines contraindicated
- absent or hypoplastic lymph organs esp. tonsils
- symptoms present after 6 months
- malignancies are rare, but more susceptible to autoimmune diseases
- treatments - Igs, antibiotics, nutritional supplements, genetic counseling
- diagnosis - serum protein electrophoresis, quantitative Igs, PHA stimulation test/candida hypersensitivity test
DiGeorge's Syndrome
- Congenital thymic dysplasia
- hypoplastic/aplastic thymus
- no T cells
- associated with other abnormalities - heart defects, cleft palate, facial abnormalities, hypocalcemia, deletion on chromosome 22
- Treatment - depends on severity of disease (antibiotics to tranplant of thymus)
Chronic Mucocutaneous Candidiasis
- Hole in repertoire
- Specific CD4 T cell subset
- Increased susceptibility to Candida infections
- Mucocutaneous candidiasis
Chronic Granulomatous Disease
- No proper function in macrophages or neutrophils
- defect in NADPH oxidase enzyme complex
- increased susceptibility to fungal infections, especially aspergillus and bacterial infections
- mycetoma
- treatment IFNg
- long term treatment - bone marrow transplant
- diagnosis - nitroblue tetrazolium test
Chediak Higashi Syndrome
- No proper function in macrophages or neutrophils
- Accumulation of large cytoplasmic granules
- Transport and fusion of lysosomes with phagosomes affected - due to defect in LYST gene
- Long term treatment bone marrow transplant
- NK cells and CTL function also affected
Leukocyte Adhesion Defects
- Affects phagocytes (neutrophils)
- Defects in b2 integrins (CD18, LAD 1)
- defect in counter molecule for E selectin (LAD 2)
- bone marrow transplant
- increased susceptibility to fungal infections, bacterial infections
Transient Hypogammaglobulinemia
- Primary immunodeficiency
- presents at 6 months
- recurrent infection with pyogenic bacteria
- treatment: gamma globulins
- improves with age
Common Varibale Hypoglammaglobulinemia
- Primary immunodeficiency
- B cell differentiation block
- onset at any age
- treatment: gamma globulins
- may develop autoantibodies
- increased susceptibility to viral and fungal infections
Selective IgA deficiency
- Primary B cell immunodeficiency
- No isotype switching to IgA
- Productions of other Abs isotypes unaffected
- manifests as chronic sinus infections
- may have transfusion reactions if anti-IgA present
- increased incidence of autoimmune conditions
Hyper IgM syndrome
- Primary B cell immunodeficiency
- Defect is CD40-CD40L interaction
- absence of isotype switching in extreme cases
- X linked disorder
- Low levels of IgG, IgA, and IgE
Complement System Related immunodeficiency
- Primary immunodeficiency
- hereditary angioedema (C1 inhibitor protein deficiency), C3 deficiency, deficiencies in C5-C9, paroxysmal nocturnal hemoglobinuria (PNH)
Hereditary Angioedema
- C1 inhibitor defect
- increased production of C2b and C4a and bradykinin
- acute, intermittent attacks of skin and mucosal edema
Paroxysmal Nocturnal Hemoglobinuria
- Changes in GPI anchored proteins (CD59/protectin, DAF/CD55), not able to be synthesized
- Susceptibility of cell membranes on erythrocytes (don't have transmembrane forms like other cells, only GPI) to lyse by complement activity, leading to hemolysis
- Symptoms: severe anemia, fatigue, pain, breathing, kidney disease, thromboses
-Treat with Anti-C5 antibody: prevention of MAC and osmolysis
Side effects: Meningococcal infections
Type I Hypersensitivity
- Systemic: Anaphylaxis, Local: Asthma or Allergic rhinits
- Involves IgE production - to allergen, usually innocuous environmental antigen
- IgE binds FceR on mast cells and basophils
- Subsequent exposure to allergen brings about crosslinkage of adjacent IgE molecules causing degranulation
- Histamine (and other slower mediators such as SRS-A) is released - increased vascular permeability, vasodilation, increased exocrine secretion, and increased bronchiolar smooth muscle constriction - symptoms of hay fever and extrinsic asthma
- systemic responses to ingested or injected allergens could cause anaphylactic shock
- skin test - wheal and flare
- Sensitization phase: B cell picks up and is presented to Th2 cell - plasma cell secretes the IgE
- Acute phase (minutes) and late phase (hours) are present
- avoidance of trigger, anti-histamines, di-sodium cromoglycate - stablizes mast cell membranes and inhibits degranulation, epinephrine - reverses anaphylactic shock, chronic topical steroid, desensitization therapy - generate high levels of IgG Abs to allergen - blocking Abs
- acute desensitization (food and drugs): incremental doses every 30 min, desensitization lost when stopped
Type II Hypersensitivity
- involves IgG or IgM Abs against membrane-bound Ag
- damage to membranes or membrane dysfunction
- examples: tranfusion rxns, HDN, Good Pasteurs, Graves, Myasthenia gravis, (autolgous cell death), chronic idiopathic urticaria
- Sensitization phase: production of IgG or IgM Abs for a specific cell surface Ag. epitope can be a hapten.
- Effector phase: as soon as Abs generated because Ag not eliminated in primary response
- Effector mechanisms: Opsonization (IgG, C3b), ADCC (NK cells with FcgR), Complement activation (C3b, MAC, C3a/C4a/C5a cause mast cell degranulation - NOT IgE)
Hemolytic Disease of the newborn Testing
Direct Coombs test/DAT: fetal RBCs coated with maternal IgG Abs. The addition of anti-Ig would cause agglutination - positive DAT.

Indirect Coombs test/IAT: mother's serum contains anti-D. When added to Rh+ RBCs, the anti-D would coat them. The addition of anti-Ig would cause agglutination - positive IAT.
Type III Hypersensitivity
Immune Complex Mediated
- Associated with IgG or IgM Abs directed against soluble antigens
- Ag and Ab form soluble immune complexes, which stimulate the complement cascade
- Sensitization phase: Production of IgM and IgG Abs to a solube Ag - persistent Ag exposure
- Effector phase: immune complexes deposited in capillary walls; complexes trapped btwn contracted endothelial cells; complement activation; frustrated phagocytosis; tissue damage leads to activation of intrinsic coagulation pathway and associated effects.
- Effector cells are neutrophils and platelets
- Pathology related to complex deposition and neutrophil mediated damage (frustrated phagocytosis)
- Examples: Arthus reaction (localized), serum sickness, farmer's lung (allergic alveolitis), SLE, rheumatoid, polyarteritis, aspergillosis
Arthus Reaction
Localized Type III phenomenon
- Ag introduced locally into an individual who has pre-formed antibodies against an Ag
- Classical time scale of damage is 5-8 hrs after exposure to Ag
Type IV Hypersensitivity
Delayed Type/Cell mediated
- Only type not transferable with serum
- Ag is usually exogenous but may be endogenous
- Ag processing leads to sensitization of memory Th1 cells, which then react on re-exposure to the Ag leading to the clinical symptoms
- Time course: 2-3 days
- Patch test
- Classification - chronic (granulomatous inflammation, acute type 4 hypersensitivity, contact hypersensitivity)
- Sensitization phase: cell mediated (not Abs), Thp cells differentiation, memory Th cells
- Effector phase: Chronic persistent IC infection (TB); re-exposure to Ag and activation of memory Th1 cells - skin introduction of Ag; tissue damage (non-specific inflammatory mediators)
- Regulation of response: Ag elimination, increase in Th2 cytokines
Examples: contact hypersensitivity/dermatitis (poison ivy), Tuberculin test/Mantoux rxn, granuloma formation
- Chronic manifestations: granulomatous inflammation (mycobacterium); tuberculoid leprosy; granuloma formation (sarcoidosis or chronic hypersensitivity pneumonitis)
- Acute manifestations: mantoux reaction (tuberculin); contact hypersensitivity (poison ivy)
Contact Hypersensitivity
- Simple foreign Ag (metal ion, chemical, plant) contacts epidermis
- Ag becomes complexed with self protein in the skin - Ag is hapten and protein is carrier
- Taken up by Langerhans cells in the skin (APCs)
- Ags presented to Th1 cells in lymph nodes
- Sensitized Th1 cells home to site of Ag deposition
- Effector Th1 cells secrete inflammatory cytokine cascade causing local tissue damage
Mantoux Reaction
- PPD or tuberculin injected into dermis
- APCs present Ag to Th1 memory cells (if present)
- If present, memory Th1 cells produce localized site of inflammation
- leads to induration and erythema at site of injection
- result read after 2-3 days
Granuloma formation
- Chronic exposure to Ag (TB, leprosy, celiac disease, chrons, sarcoidosis, foreign body)
- Th1 effector cells secrete proinflammatory cytokines and chemokines directed at macrophages
- Granulomas form to wall off antigen
- Cause tissue dysfunction and disfigurement
Tumor recognition
1. Tumor specific antigens (unique to cancer cells) - potential therapeutic target. The concept of aiming the magic bullet
2. Tumor associated antigens - normally only expressed in development (embryo, fetus)
3. Over-expression of normal self proteins
Tumor evasion method
1. Antigen loss - variant of tumor cell
2. Class I MHC deficient tumor cell/mutated MHC I (but more susceptible to NK cells)
3. Production of immunosuppressive molecules
DNA Vaccines
In response to the fact that delivery of exogenous proteins will not produce an MHC I response (no activation of CTL), which is important in viral diseases
- Naked DNA plasmid contains sequence that codes for an antigen or immunogenic epitope
- It has a promotor region for more efficient transcription
- Elicits both a cell mediated and humoral immune response
- Under investigation for HIV/AIDS, infuenza, malaria, Hep B
Paroxysomal Nocturnuria
- Changes in GPI anchored proteins (CD59, DAF/CD55), not able to be synthesized
- Susceptibility of cell membranes on erythrocytes (don't have transmembrane forms like other cells, only GPI) to lyse by complement activity, leading to hemolysis
- Symptoms: severe anemia, fatigue, pain, breathing, kidney disease, thromboses
-Treat with Anti-C5 antibody: prevention of MAC and osmolysis
Side effects: Meningococcal infections
Allergic Rhinitis
- Aka Hay fever, nasal allergies
- Type 1 Hypersensitivty
- Seasonal or perennial
- Associated with eczema, asthma, food allergy
- certain climates and environments may be triggers - mold, pollen, dust, mites, dander
- Symptoms: runny nose, cough, itchy eyes, red throat, sneezing, headache, nasal congestion, drowsiness, postnasal drip, blockage of eustachian tube is possible
- Cromolyn Sodium - mast cell stabilizer
- Anti-histamines
- intranasal steroids - used when symptoms not relieved with AH or if congestion is prominent
- Leukotriene receptor antagonists