Immunology

1. Overview of the Immune System

The immune system is a network of cells, tissues, and organs that defends the body against pathogens (bacteria, viruses, fungi, protozoa) and other harmful agents. It is divided into two complementary branches:

Feature	Innate Immunity	Adaptive (Acquired) Immunity
Specificity	Non-specific: responds to broad categories of PAMPs	Highly specific: recognises individual antigens
Response time	Immediate (minutes to hours)	Delayed (days): requires clonal selection and expansion
Memory	No immunological memory	Memory cells provide faster, stronger secondary response
Components	Physical barriers, phagocytes, complement, NK cells	T lymphocytes, B lymphocytes, antibodies
Receptors	Fixed, germline-encoded pattern recognition receptors	Diverse receptors generated by V(D)J recombination

The two systems are tightly integrated: innate immunity provides the initial defence and activates adaptive immunity through antigen presentation; adaptive immunity provides specific, long-lasting protection and enhances innate responses through antibody opsonisation and cytokine secretion.

2. Innate Immunity

First-Line Defences (Physical and Chemical Barriers)

These are always present and prevent pathogen entry.

Barrier	Mechanism
Skin	Physical barrier; keratinised outer layer; sebum (antimicrobial lipids); low pH ( $\approx 5.5$ ); normal flora compete with pathogens.
Mucous membranes	Trap pathogens in sticky mucus; ciliated epithelium moves mucus upward (mucociliary escalator in trachea).
Lysozyme	Enzyme in tears, saliva, and mucus that hydrolyses peptidoglycan in bacterial cell walls, causing lysis.
Stomach acid	$\mathrm{HCl}$ (pH $\approx 1.5$ -- $2.0$ ) destroys most ingested pathogens.
Normal flora	Commensal bacteria on skin and in the gut outcompete pathogens for nutrients and binding sites; produce antimicrobial substances.

Second-Line Defences (Internal Non-Specific Responses)

Phagocytosis

Phagocytes (neutrophils, macrophages, and dendritic cells) engulf and destroy pathogens.

Steps of phagocytosis:

Chemotaxis: phagocytes migrate toward chemical attractants released at the infection site (e.g., bacterial peptides, complement fragments $\mathrm{C3a}$ , $\mathrm{C5a}$ ).
Recognition: phagocyte binds to pathogen via:
- Direct binding to PAMPs (pathogen-associated molecular patterns) using pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs).
- Indirect binding via opsonins (antibodies or complement $\mathrm{C3b}$ ) coating the pathogen surface.
Engulfment: pseudopodia extend around the pathogen, enclosing it in a membrane-bound vesicle called a phagosome.
Digestion: the phagosome fuses with a lysosome, forming a phagolysosome. Hydrolytic enzymes (proteases, lipases, nucleases, lysozyme) and reactive oxygen species ( $\mathrm{H}_2\mathrm{O}_2$ , superoxide radicals) degrade the pathogen.
Exocytosis: indigestible material is expelled from the cell.
Antigen presentation: peptide fragments of the digested pathogen are displayed on MHC molecules on the phagocyte surface, initiating the adaptive immune response (see Section 4).

Neutrophils are short-lived (circulate for $6$ -- $10$ hours), abundant, and are the first phagocytes to arrive at infection sites. Macrophages are long-lived, more versatile, and play a central role in antigen presentation.

Inflammation

Inflammation is a localised tissue response to damage or infection, characterised by the four cardinal signs: rubor (redness), calor (heat), tumor (swelling), and dolor (pain).

Mechanism:

Mast cells and damaged tissues release histamine and other inflammatory mediators.
Histamine causes vasodilation (increased blood flow, producing redness and heat) and increases capillary permeability (plasma leaks into tissues, causing swelling and pain).
Increased blood flow delivers more phagocytes (neutrophils first, then macrophages) to the site.
Fibrinogen leaks into the tissue and is converted to fibrin, forming a clot that walls off the infected area.
Macrophages release cytokines (e.g., interleukin-1, tumour necrosis factor-alpha) that promote inflammation and fever.

The Complement System

A cascade of plasma proteins ( $\mathrm{C1}$ through $\mathrm{C9}$ ) that enhances immune defence through three pathways:

Pathway	Trigger
Classical pathway	Antibody-antigen complexes (adaptive immunity)
Lectin pathway	Mannose-binding lectin binding to pathogen carbohydrates
Alternative pathway	Spontaneous hydrolysis of $\mathrm{C3}$ on microbial surfaces

All three pathways converge on $\mathrm{C3}$ and lead to:

Opsonisation ( $\mathrm{C3b}$ ): coats pathogens, enhancing phagocytosis.
Inflammation ( $\mathrm{C3a}$ , $\mathrm{C5a}$ ): act as chemoattractants for phagocytes; cause mast cell degranulation.
Membrane attack complex (MAC) ( $\mathrm{C5b\text{-}C9}$ ): forms transmembrane pores in the pathogen cell membrane, causing lysis by osmotic imbalance.

Natural Killer (NK) Cells

NK cells are large granular lymphocytes that detect and kill virus-infected cells and cancerous cells. They recognise cells that have:

Downregulated MHC class I expression (common in virus-infected and tumour cells --- "missing self").
Stress-induced ligands (e.g., MICA, MICB) on their surface.

NK cells kill by releasing perforin (forms pores) and granzymes (induce apoptosis), a mechanism similar to cytotoxic T cells but without prior sensitisation.

Interferons

Interferons are cytokines produced by virus-infected cells that signal neighbouring cells to:

Produce antiviral proteins that inhibit viral replication (e.g., protein kinase R, which phosphorylates and inactivates eukaryotic initiation factor eIF-2 $\alpha$ , blocking viral protein synthesis).
Upregulate MHC class I expression, enhancing antigen presentation to cytotoxic T cells.
Activate NK cells.

Type I interferons (IFN- $\alpha$ and IFN- $\beta$ ) are produced by most nucleated cells. Type II interferon (IFN- $\gamma$ ) is produced by T cells and NK cells and activates macrophages.

3. Adaptive Immunity: Overview

Adaptive immunity relies on lymphocytes (T cells and B cells) that express highly specific antigen receptors. The diversity of these receptors is generated by V(D)J recombination during lymphocyte development in the bone marrow (B cells) and thymus (T cells).

Lymphocyte development:

B cells: mature in the bone marrow. Each B cell expresses a unique B-cell receptor (BCR) (membrane-bound antibody) specific for one antigen.
T cells: produced in the bone marrow but migrate to the thymus to mature. Each T cell expresses a unique T-cell receptor (TCR) specific for one antigen-MHC complex.

Clonal selection: when a lymphocyte encounters its specific antigen, it is activated, proliferates (clonal expansion), and differentiates into effector cells and memory cells.

4. Antigen Presentation

Antigen-presenting cells (APCs --- dendritic cells, macrophages, and B cells) process and display antigen fragments on MHC molecules for T cell recognition.

MHC Class I (Endogenous Pathway)

Found on all nucleated cells.
Presents endogenous antigens: peptide fragments from proteins synthesised within the cell (e.g., viral proteins in an infected cell, tumour antigens).
Recognised by $\mathrm{CD8}^+$ cytotoxic T cells.

Processing:

Intracellular proteins are degraded by the proteasome into peptide fragments.
Peptides are transported into the endoplasmic reticulum (ER) by TAP (transporter associated with antigen processing).
Peptides bind to MHC class I molecules in the ER.
The MHC I--peptide complex is transported to the cell surface via the Golgi apparatus.

MHC Class II (Exogenous Pathway)

Found only on professional APCs (dendritic cells, macrophages, B cells).
Presents exogenous antigens: peptide fragments from pathogens engulfed by phagocytosis.
Recognised by $\mathrm{CD4}^+$ helper T cells.

Processing:

APC engulfs a pathogen by phagocytosis, forming a phagosome.
The phagosome fuses with a lysosome; hydrolytic enzymes degrade the pathogen into peptides.
MHC class II molecules (synthesised in the ER with an invariant chain blocking the peptide-binding groove) are transported to the endosome.
The invariant chain is degraded; peptides from the pathogen bind to the MHC class II groove.
The MHC II--peptide complex is transported to the cell surface.

5. T-Cell Activation and Response

Helper T Cells ( $\mathrm{CD4}^+$ )

Helper T cells orchestrate the adaptive immune response through cytokine secretion.

Activation (two-signal model):

Signal 1: TCR binds to antigen-MHC class II complex on an APC.
Signal 2 (co-stimulation): $\mathrm{CD28}$ on the T cell binds to $\mathrm{B7}$ ( $\mathrm{CD80/CD86}$ ) on the APC. Without co-stimulation, the T cell becomes anergised (unresponsive) --- this prevents autoimmunity.
The activated helper T cell undergoes clonal expansion and differentiates into subsets based on cytokine signals:

Subset	Cytokines driving differentiation	Key cytokines produced	Primary function
$\mathrm{T_H}1$	IL-12	IFN- $\gamma$ , IL-2	Activates macrophages (cell-mediated immunity against intracellular pathogens).
$\mathrm{T_H}2$	IL-4	IL-4, IL-5, IL-6, IL-10	Stimulates B cell activation and antibody production (humoral immunity against extracellular pathogens).
$\mathrm{T_H}17$	TGF- $\beta$ + IL-6	IL-17, IL-22	Recruits neutrophils; defends against extracellular bacteria and fungi.
$\mathrm{T_{reg}}$	TGF- $\beta$ + IL-2	TGF- $\beta$ , IL-10	Suppresses immune responses; maintains self-tolerance.

Cytotoxic T Cells ( $\mathrm{CD8}^+$ )

Cytotoxic T cells (CTLs) directly kill infected or cancerous cells.

Activation:

TCR binds to antigen-MHC class I on a target cell.
Co-stimulation (often provided by helper T cell cytokines, especially IL-2).
CTL undergoes clonal expansion and differentiation.

Killing mechanism:

The CTL releases perforin, which polymerises in the target cell membrane to form transmembrane pores.
Granzymes (serine proteases) enter through the pores and activate caspases, triggering apoptosis (programmed cell death).
The target cell fragments into apoptotic bodies that are phagocytosed by macrophages.

Advantages of apoptosis over necrosis: pathogen contents are contained within apoptotic bodies, preventing release of infectious particles; no inflammatory response is triggered.

Memory T Cells

After an infection is cleared, a subset of T cells differentiates into long-lived memory T cells:

Central memory T cells ( $\mathrm{T_{CM}}$ ): reside in lymphoid organs; rapidly proliferate upon re-exposure.
Effector memory T cells ( $\mathrm{T_{EM}}$ ): patrol peripheral tissues; provide immediate defence at the site of re-infection.

Memory T cells enable a faster, stronger secondary immune response upon re-exposure to the same pathogen.

6. B-Cell Activation and Humoral Immunity

B-Cell Activation

T-dependent activation (requires helper T cells; most protein antigens):

B cell binds free antigen via its surface BCR (membrane-bound immunoglobulin).
The B cell internalises the antigen, processes it, and presents antigen peptides on MHC class II.
A helper T cell with a matching TCR recognises the antigen-MHC II complex on the B cell surface (two-signal model: TCR-MHC II interaction + CD40-CD40L co-stimulation).
The helper T cell secretes cytokines (IL-4, IL-5, IL-6) that stimulate the B cell to proliferate and differentiate.

T-independent activation (no helper T cells required; polysaccharide and LPS antigens):

The antigen has repetitive epitopes that cross-link multiple BCRs on the B cell surface, providing sufficient signal for activation.
Produces mainly IgM antibodies; no affinity maturation or class switching; limited memory.

B-Cell Differentiation

Clonal expansion: activated B cells proliferate.
Plasma cells: short-lived (3--5 days) effector cells that secrete large quantities of antibodies ( $\approx 1000$ antibody molecules per second).
Memory B cells: long-lived cells that provide rapid secondary responses upon re-exposure.

Affinity Maturation and Class Switching

These processes occur in the germinal centres of lymph nodes during T-dependent responses:

Somatic hypermutation: B cells in the germinal centre introduce point mutations in the variable regions of their BCR genes at a rate $\approx 10^6$ times higher than the normal mutation rate. B cells with higher-affinity BCRs are positively selected (receive survival signals from follicular helper T cells), while lower-affinity cells undergo apoptosis.

Class (isotype) switching: cytokines from helper T cells direct the B cell to switch from producing IgM to other antibody classes (IgG, IgA, IgE) by changing the constant region of the heavy chain while preserving the antigen-specific variable region. The DNA encoding the constant region is excised by activation-induced cytidine deaminase (AID).

7. Antibody Structure and Classes

Antibody (Immunoglobulin) Structure

Antibodies are Y-shaped glycoproteins composed of:

Two identical heavy chains ( $\approx 50\;\mathrm{kDa}$ each)
Two identical light chains ( $\approx 25\;\mathrm{kDa}$ each)

Connected by disulfide bonds. Each chain contains:

Variable ( $V$ ) regions: at the N-terminus, unique to each B cell clone. The antigen-binding site is formed by the variable regions of one heavy and one light chain ( $V_H$ and $V_L$ ), creating a paratope complementary to the antigen's epitope.
Constant ( $C$ ) regions: determine the antibody class (isotype) and effector functions.

Each antibody has two identical antigen-binding sites (bivalent), allowing cross-linking of antigens.

Hinge region: flexible region between the two Fab arms and the Fc stem, allowing the antibody to bind to antigens at varying distances. The hinge region varies between antibody classes.

Antibody Classes

Class	Heavy chain	Structure	Location	Function	Percentage of serum antibodies
IgG	$\gamma$	Monomer	Blood, lymph, interstitial fluid	Most abundant; opsonisation; complement activation (classical pathway); crosses placenta (passive immunity to foetus); neutralisation.	$\approx 75\%$
IgA	$\alpha$	Monomer (blood) or dimer (mucosa)	Mucosal surfaces, saliva, tears, breast milk, GI tract	Prevents pathogen attachment to epithelial surfaces; dimeric form has a secretory component protecting it from proteolysis.	$\approx 15\%$
IgM	$\mu$	Pentamer (10 binding sites)	Blood (first antibody produced)	First antibody produced in primary response; very effective at agglutination and complement activation; monomeric form serves as BCR.	$\approx 10\%$
IgE	$\epsilon$	Monomer	Bound to mast cells and basophils	Allergic responses; anti-parasitic (helminth) defence; triggers mast cell degranulation (histamine release).	$< 0.01\%$
IgD	$\delta$	Monomer	Surface of naive B cells	Functions as BCR alongside IgM; exact function not fully understood.	Trace

Antibody Functions (Effector Mechanisms)

Neutralisation: antibodies bind to pathogen surface proteins or toxin active sites, blocking their ability to enter host cells or cause damage. Example: neutralising antibodies against the SARS-CoV-2 spike protein prevent viral entry.
Opsonisation: the Fc region of IgG antibodies is recognised by $\mathrm{Fc\gamma}$ receptors on phagocytes, enhancing phagocytosis. $\mathrm{C3b}$ complement fragments also serve as opsonins.
Agglutination: bivalent (or multivalent) antibodies cross-link pathogens into large clumps that are more easily phagocytosed and physically immobilised. IgM is particularly effective due to its 10 binding sites.
Complement activation: antigen-antibody complexes activate the classical complement pathway ( $\mathrm{C1q}$ binds to the Fc region), leading to opsonisation, inflammation, and MAC-mediated lysis.
Antibody-dependent cell-mediated cytotoxicity (ADCC): NK cells recognise antibody-coated target cells via $\mathrm{Fc\gamma}$ receptors and kill the target cell by releasing perforin and granzymes.
Mucosal immunity (IgA): secretory IgA in mucus traps pathogens and prevents their adhesion to epithelial cells, providing a first line of immune defence at mucosal surfaces.

8. Immunological Memory

Primary and Secondary Responses

Feature	Primary Response	Secondary Response
Trigger	First exposure to antigen	Re-exposure to the same antigen
Lag phase	$5$ -- $10$ days (clonal selection and expansion)	$1$ -- $3$ days (memory cells already present)
Antibody titre	Low (peak IgM, then IgG)	High (rapid IgG production; peak titre $10$ -- $100\times$ higher)
Antibody affinity	Lower (no somatic hypermutation initially)	Higher (affinity maturation has occurred)
Antibody class	IgM predominates initially, then class switching	IgG predominates (memory B cells already class-switched)
Duration	Short-lived (effector cells die after clearance)	Long-lived (memory cells persist for years to decades)

Types of Immunity

Type	Description	Example
Active natural	Immunity acquired after natural infection; memory cells generated.	Chickenpox immunity after infection.
Active artificial	Immunity acquired after vaccination; antigen is introduced without disease.	MMR vaccine immunity.
Passive natural	Antibodies transferred from mother to foetus (IgG across placenta) or newborn (IgA in colostrum).	Newborn immunity to maternal infections.
Passive artificial	Pre-formed antibodies administered (e.g., antivenom, monoclonal antibodies).	Rabies immunoglobulin after potential rabies exposure.

9. Vaccination

Principles

Vaccines expose the immune system to a pathogen antigen (or a weakened/inactivated pathogen) to stimulate a primary response and generate memory cells without causing disease. Upon subsequent exposure, the secondary response provides rapid, effective protection.

Types of Vaccines

Type	Description	Example
Live attenuated	Weakened (attenuated) live pathogen; replicates within the host, stimulating strong immunity.	MMR (measles, mumps, rubella); BCG (TB)
Inactivated (killed)	Pathogen killed by heat or chemicals; cannot replicate; generally requires booster doses.	Influenza (injected); cholera; hepatitis A
Toxoid	Inactivated bacterial toxin; induces antibodies that neutralise the toxin.	Tetanus; diphtheria
Subunit (recombinant)	Purified antigenic components (proteins or polysaccharides) from the pathogen.	HPV (virus-like particles); hepatitis B
Conjugate	Polysaccharide antigen linked to a carrier protein; improves immune response in young children.	Pneumococcal; meningococcal
mRNA	Messenger RNA encoding a pathogen antigen; host cells translate the mRNA to produce the antigen.	Pfizer/BioNTech and Moderna COVID-19 vaccines
Viral vector	Harmless virus engineered to carry genetic material encoding a pathogen antigen.	Oxford/AstraZeneca COVID-19 vaccine; Ebola

Vaccine Considerations

Herd immunity: when a sufficient proportion of the population is immune ( $> 90\%$ for measles, $\approx 80\%$ for polio), transmission is interrupted, protecting unvaccinated individuals. Herd immunity threshold: $R_0 - 1 / R_0$ where $R_0$ is the basic reproduction number.
Antigenic drift: minor mutations in surface proteins (e.g., influenza haemagglutinin) require annual vaccine updates. Antigenic shift: major genetic reassortment (influenza) can produce pandemic strains against which existing immunity is ineffective.
Vaccine failures: primary failure (no immune response generated) and secondary failure (waning immunity over time). Booster doses address secondary failure.

10. ELISA (Enzyme-Linked Immunosorbent Assay)

ELISA is a quantitative immunological assay used to detect and measure the concentration of antigens or antibodies in a sample.

Direct ELISA (detecting antigen)

Antigen is immobilised on the wells of a microtitre plate.
A specific primary antibody (conjugated to an enzyme) is added and binds to the antigen.
The plate is washed to remove unbound antibody.
A substrate is added; the enzyme catalyses a colour change.
The intensity of colour (measured by spectrophotometer) is proportional to the amount of antigen present.

Indirect ELISA (detecting antibody --- e.g., HIV testing)

Antigen is immobilised on the plate.
The patient's serum (containing unknown antibodies) is added. If antibodies are present, they bind to the antigen.
The plate is washed.
An enzyme-conjugated secondary antibody (anti-human immunoglobulin) is added, binding to any patient antibodies present.
Substrate is added; colour change indicates the presence of patient antibodies.

Sandwich ELISA (detecting antigen with higher sensitivity)

A capture antibody specific to the antigen is immobilised on the plate.
The sample (containing unknown antigen) is added; antigen is captured.
A detection antibody (enzyme-conjugated) binds to a different epitope on the captured antigen, forming a "sandwich."
Substrate is added; colour intensity is proportional to antigen concentration.

Common enzyme-substrate pairs: horseradish peroxidase (HRP) + TMB (produces blue colour, turns yellow with acid stop solution); alkaline phosphatase (AP) + p-nitrophenyl phosphate (PNPP, produces yellow colour).

11. Monoclonal Antibodies

Production (Kohler and Milstein, 1975)

A mouse is injected with a target antigen, stimulating B cell production.
B cells from the mouse spleen are harvested.
These B cells are fused with myeloma cells (immortal cancer cells) using polyethylene glycol (PEG) or electrofusion.
The resulting hybridomas combine the antibody-producing capability of B cells with the immortality of myeloma cells.
Hybridomas are cultured in HAT medium (hypoxanthine-aminopterin-thymidine): unfused myeloma cells die (blocked de novo nucleotide synthesis), unfused B cells die (short-lived); only hybridomas survive.
Individual hybridomas are screened for the desired antibody specificity.
A single hybridoma clone is expanded; it produces a single type of antibody indefinitely (monoclonal = "one clone").

Applications

Application	Description
Medical diagnosis	Pregnancy tests (detect hCG); detecting cancer antigens (e.g., PSA for prostate cancer); detecting pathogen antigens in ELISA.
Cancer treatment	Trastuzumab (Herceptin) targets HER2 receptor on breast cancer cells; rituximab targets $\mathrm{CD20}$ on B-cell lymphomas.
Autoimmune diseases	Infliximab targets TNF- $\alpha$ in rheumatoid arthritis and Crohn's disease.
Drug delivery	Monoclonal antibodies conjugated to drugs or radioactive isotopes deliver therapy specifically to cancer cells, minimising side effects.
COVID-19 treatment	Monoclonal antibody cocktails (e.g., REGEN-COV) were used as passive immunotherapy for high-risk patients.

12. Organ Transplantation

Types of Transplants

Type	Description
Autograft	Tissue transplanted from one site to another in the same individual.
Isograft	Transplant between genetically identical individuals (identical twins).
Allograft	Transplant between genetically non-identical members of the same species (most common type).
Xenograft	Transplant from a different species (e.g., pig heart valve).

Transplant Rejection

The immune system recognises foreign MHC molecules (also called HLA --- human leucocyte antigen in humans) on transplanted tissue as non-self, mounting an immune response.

Type of rejection	Timing	Mechanism
Hyperacute	Minutes to hours	Pre-existing anti-donor antibodies (from prior exposure, blood transfusion, pregnancy) bind to donor endothelium, activating complement and causing thrombosis.
Acute	Days to weeks	T cell-mediated rejection: $\mathrm{CD8}^+$ cytotoxic T cells kill donor cells expressing foreign MHC I; $\mathrm{CD4}^+$ helper T cells activate macrophages and B cells.
Chronic	Months to years	Chronic inflammation and fibrosis; both T-cell and antibody-mediated damage; leading cause of late graft failure.

Immunosuppression

Transplant recipients receive immunosuppressive drugs to prevent rejection:

Calcineurin inhibitors: cyclosporin A, tacrolimus --- block T cell activation by inhibiting IL-2 transcription.
Corticosteroids: prednisolone --- suppress inflammation and immune cell proliferation.
Antimetabolites: azathioprine, mycophenolate mofetil --- inhibit DNA synthesis in dividing lymphocytes.
mTOR inhibitors: sirolimus (rapamycin) --- inhibit T cell proliferation.

Complications of immunosuppression: increased susceptibility to infections, increased cancer risk (especially lymphomas and skin cancers), nephrotoxicity (calcineurin inhibitors).

Tissue Typing and Cross-Matching

HLA typing: the donor's and recipient's HLA alleles are matched to minimise rejection risk. The major HLA loci are HLA-A, HLA-B, and HLA-DR.
ABO blood group matching: ABO antigens are expressed on endothelial cells; ABO incompatibility causes hyperacute rejection.
Cross-match: recipient serum is mixed with donor lymphocytes to detect pre-existing anti-donor antibodies.

13. HIV/AIDS

HIV Structure and Life Cycle

Human immunodeficiency virus (HIV) is a retrovirus:

RNA genome (two copies of positive-sense ssRNA).
Envelope: derived from the host cell membrane, studded with viral glycoproteins gp120 (binds $\mathrm{CD4}$ ) and gp41 (mediates membrane fusion).
Capsid: protein shell enclosing the RNA genome and viral enzymes.
Enzymes: reverse transcriptase (RNA $\to$ DNA), integrase (inserts viral DNA into host genome), protease (cleaves viral polyproteins into functional proteins).

Life cycle:

Attachment: gp120 on HIV binds to $\mathrm{CD4}$ receptor and a co-receptor ( $\mathrm{CCR5}$ or $\mathrm{CXCR4}$ ) on helper T cells, macrophages, and dendritic cells.
Entry: gp41 mediates fusion of the viral envelope with the host cell membrane; viral contents enter the cell.
Reverse transcription: reverse transcriptase converts viral RNA into double-stranded DNA.
Integration: integrase inserts the viral DNA (provirus) into the host cell's genome.
Latency: the provirus may remain dormant for years, invisible to the immune system.
Activation: when the host cell is activated, viral genes are transcribed and translated.
Assembly and budding: new viral particles assemble at the cell membrane and bud off, acquiring an envelope.

Immunological Effects

HIV selectively infects and destroys $\mathrm{CD4}^+$ helper T cells, which are central to coordinating both humoral and cell-mediated immunity.
Progressive depletion of helper T cells weakens the immune system, leading to acquired immunodeficiency syndrome (AIDS).
AIDS is defined by $\mathrm{CD4}^+$ T cell count below $200\;\mathrm{cells/\mu L}$ (normal: $500$ -- $1500\;\mathrm{cells/\mu L}$ ) and/or the presence of opportunistic infections (e.g., Pneumocystis jirovecii pneumonia, Mycobacterium avium complex, Kaposi's sarcoma caused by HHV-8, candidiasis, tuberculosis).

Transmission and Prevention

Route	Prevention strategies
Sexual contact	Condoms; pre-exposure prophylaxis (PrEP); treatment as prevention (TasP --- viral suppression reduces transmission).
Blood (transfusion, needles)	Screening donated blood; needle exchange programmes; sterile equipment.
Mother-to-child	Antiretroviral therapy for mother during pregnancy and delivery; caesarean section; avoidance of breastfeeding.

Antiretroviral Therapy (ART)

Standard treatment: combination antiretroviral therapy (cART) using three or more drugs from at least two classes:

Drug class	Target	Example drugs
Nucleoside reverse transcriptase inhibitors (NRTIs)	Competitive inhibition of reverse transcriptase	Zidovudine (AZT), lamivudine (3TC)
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)	Allosteric inhibition of reverse transcriptase	Efavirenz, nevirapine
Protease inhibitors (PIs)	Block viral protease	Ritonavir, lopinavir
Integrase strand transfer inhibitors (INSTIs)	Block integrase	Dolutegravir, raltegravir
Entry inhibitors	Block CCR5 co-receptor (maraviroc) or gp41 fusion (enfuvirtide)	Maraviroc, enfuvirtide

ART suppresses viral replication to undetectable levels ( $< 50$ copies/mL), restoring immune function but not eradicating the virus (latent proviruses persist in reservoirs).

14. Autoimmune Diseases

Autoimmune diseases arise when the immune system fails to distinguish self from non-self and attacks the body's own tissues.

Mechanisms of Loss of Self-Tolerance

Molecular mimicry: a pathogen antigen shares structural similarity with a self-antigen; immune cells activated against the pathogen cross-react with self-tissue. Example: Streptococcus pyogenes M protein resembles cardiac myosin, leading to rheumatic heart disease.
Failure of central tolerance: autoreactive T cells escape deletion in the thymus.
Failure of peripheral tolerance: regulatory T cells ( $\mathrm{T_{reg}}$ ) fail to suppress autoreactive lymphocytes.
Epitope spreading: tissue damage releases new self-antigens, broadening the immune response.
Polyclonal activation: superantigens (e.g., staphylococcal enterotoxins) non-specifically activate large numbers of T cells, including autoreactive clones.

Examples of Autoimmune Diseases

Disease	Target tissue	Mechanism
Type 1 diabetes mellitus	Pancreatic beta cells	$\mathrm{CD8}^+$ cytotoxic T cells destroy insulin-producing beta cells.
Multiple sclerosis (MS)	Myelin sheath of neurons	T cells and antibodies attack myelin; demyelination disrupts nerve conduction.
Rheumatoid arthritis (RA)	Synovial joints	Autoantibodies (rheumatoid factor, anti-CCP) form immune complexes in joints; T cell-mediated inflammation.
Systemic lupus erythematosus (SLE)	Widespread (nuclei, blood cells, kidneys)	Antinuclear antibodies (ANA) form immune complexes deposited in multiple organs.
Myasthenia gravis	Acetylcholine receptors at neuromuscular junction	Autoantibodies block or destroy ACh receptors, causing muscle weakness.
Graves' disease	TSH receptor on thyroid	Autoantibodies stimulate the TSH receptor, causing hyperthyroidism.

15. Allergies (Type I Hypersensitivity)

Allergies are exaggerated IgE-mediated immune responses to harmless environmental antigens (allergens) such as pollen, dust mite faeces, animal dander, and food proteins.

Mechanism (Sensitisation and Challenge)

Sensitisation (first exposure):

Allergen is processed by APCs and presented to $\mathrm{T_H}2$ cells.
$\mathrm{T_H}2$ cells release IL-4 and IL-13, stimulating B cells to class-switch to IgE.
IgE antibodies bind to $\mathrm{Fc\varepsilon}$ receptors on mast cells and basophils (sensitisation).

Challenge (subsequent exposure):

The allergen cross-links IgE molecules on the surface of sensitised mast cells.
Mast cells degranulate, releasing:
- Histamine: vasodilation, increased capillary permeability, bronchoconstriction, mucus production.
- Heparin: anticoagulant.
- Proteases: tissue damage.
Late-phase reaction ( $4$ -- $12$ hours): cytokines (IL-4, IL-5, IL-13) recruit eosinophils, which release inflammatory mediators, causing sustained inflammation.

Symptoms

System	Symptoms	Severe form
Respiratory	Rhinitis (sneezing, runny nose); asthma (bronchoconstriction)	Anaphylactic shock (airway obstruction)
Skin	Urticaria (hives); eczema; angioedema (swelling)	---
Gastrointestinal	Nausea, vomiting, diarrhoea	---
Cardiovascular	Vasodilation, drop in blood pressure	Anaphylactic shock (circulatory collapse)

Anaphylaxis

A severe, potentially fatal systemic allergic reaction:

Rapid onset (minutes): airway oedema, bronchospasm, hypotension, urticaria.
Treatment: intramuscular adrenaline (epinephrine) --- constricts blood vessels (increases blood pressure), relaxes bronchial smooth muscle (relieves bronchospasm), and stabilises mast cells (reduces further degranulation).
Follow-up: antihistamines, corticosteroids, and monitoring.

Desensitisation Therapy (Allergen Immunotherapy)

Gradual, controlled exposure to increasing doses of allergen over months to years. This shifts the immune response from $\mathrm{T_H}2$ (IgE-mediated) to $\mathrm{T_H}1$ (IgG-mediated), inducing tolerance.

16. Other Immune Disorders

Immunodeficiency

Type	Cause	Example
Primary	Genetic defects in immune system components	SCID (no functional T and B cells); DiGeorge syndrome (no thymus); X-linked agammaglobulinaemia (no B cells).
Secondary	Acquired factors (infection, malnutrition, drugs, ageing, stress)	HIV/AIDS; chemotherapy-induced immunosuppression; malnutrition (zinc, vitamin A deficiency).

Type II Hypersensitivity (Antibody-Mediated)

IgG or IgM antibodies bind to antigens on cell surfaces or in the extracellular matrix, leading to:

Complement-mediated cell lysis (e.g., autoimmune haemolytic anaemia).
Antibody-dependent cell-mediated cytotoxicity (ADCC).
Opsonisation and phagocytosis (e.g., Goodpasture's syndrome --- anti-basement membrane antibodies in kidneys and lungs).

Type III Hypersensitivity (Immune Complex-Mediated)

Antigen-antibody complexes (IgG or IgM) deposit in tissues (blood vessels, joints, kidneys), activating complement and recruiting neutrophils. Neutrophil release of lysosomal enzymes causes tissue damage.

Example: serum sickness (after administration of antivenom or certain drugs); systemic lupus erythematosus (SLE).

Type IV Hypersensitivity (Delayed-Type, T-Cell Mediated)

Sensitised $\mathrm{CD4}^+$ helper T cells release cytokines (IFN- $\gamma$ , TNF- $\alpha$ ) upon re-exposure to the antigen, activating macrophages and causing inflammation. Onset: $24$ -- $72$ hours.

Examples:

Tuberculin (Mantoux) test: intradermal injection of tuberculin protein; induration indicates prior exposure to Mycobacterium tuberculosis.
Contact dermatitis: skin reaction to nickel, poison ivy (urushiol), or latex.
Type 1 diabetes: T cell-mediated destruction of pancreatic beta cells.

Common Pitfalls

Confusing antigens and antibodies: antigens are molecules that trigger an immune response (found on pathogens, transplanted tissue, or allergens); antibodies are proteins produced by plasma cells in response to antigens.
Stating that "B cells directly kill infected cells": B cells produce antibodies; cytotoxic T cells and NK cells kill infected cells directly.
Confusing IgG and IgM in primary vs secondary responses: IgM is the first antibody produced in the primary response; IgG dominates the secondary response.
Describing HIV as "killing all white blood cells": HIV specifically targets $\mathrm{CD4}^+$ helper T cells, not all leucocytes. The immunodeficiency results from the loss of T cell help, which impairs the entire adaptive immune response.
Confusing autoimmunity and immunodeficiency: autoimmunity is an overactive immune response against self-tissues; immunodeficiency is a weakened or absent immune response.
Assuming "all allergies are IgE-mediated": while Type I hypersensitivity (allergies) is IgE-mediated, other hypersensitivity types (II, III, IV) involve different mechanisms and antibody classes.
Stating that "vaccines give you the disease": live attenuated vaccines contain weakened pathogens that replicate without causing disease; inactivated, subunit, and mRNA vaccines contain no replicating pathogen at all.

Practice Problems

Question 1: ELISA Data Interpretation

An indirect ELISA is used to test four patient serum samples for antibodies against a specific virus. The absorbance readings at $450\;\mathrm{nm}$ are: Patient A = $0.05$ , Patient B = $1.85$ , Patient C = $0.92$ , Patient D = $2.10$ . The positive control gives $1.95$ and the negative control gives $0.08$ . The diagnostic threshold is an absorbance value of $0.50$ . (a) Which patients test positive? (b) Explain why a positive control and negative control are essential. (c) Why might Patient C's value be lower than Patient B's despite both being positive?

Answer

(a) Patients B ( $1.85$ ), C ( $0.92$ ), and D ( $2.10$ ) all exceed the threshold of $0.50$ and test positive. Patient A ( $0.05$ ) tests negative (consistent with the negative control).

(b) The positive control confirms the assay is working correctly (antibody is detected when known to be present). The negative control (no antibody) establishes the baseline absorbance and identifies any non-specific background signal. Without controls, false positives or false negatives cannot be identified.

(c) Patient C may have a lower antibody titre than Patient B (earlier stage of infection, weaker immune response, or immunosuppression). The absorbance is proportional to the amount of antibody bound, so a lower reading indicates less antibody in the sample. Patient C may also be in the early phase of seroconversion or have a partially effective immune response.

Question 2: HIV and T-Cell Counts

A patient is diagnosed with HIV. Their initial $\mathrm{CD4}^+$ T cell count is $550\;\mathrm{cells/\mu L}$ and their viral load is $50000\;\mathrm{copies/mL}$ . After 6 months of antiretroviral therapy, the $\mathrm{CD4}^+$ count rises to $750\;\mathrm{cells/\mu L}$ and the viral load drops to $< 50\;\mathrm{copies/mL}$ . (a) Explain the relationship between viral load and $\mathrm{CD4}^+$ T cell count. (b) Why can the virus not be completely eliminated by ART? (c) Explain why the patient is still infectious despite having an undetectable viral load.

Answer

(a) HIV infects and destroys $\mathrm{CD4}^+$ helper T cells during its replication cycle. A high viral load indicates active replication, which leads to rapid $\mathrm{CD4}^+$ depletion. ART suppresses viral replication, reducing the viral load and allowing $\mathrm{CD4}^+$ T cells to recover (the observed increase from $550$ to $750\;\mathrm{cells/\mu L}$ ).

(b) ART drugs inhibit active steps in the viral life cycle (reverse transcription, integration, protease processing), but they cannot eliminate cells containing latent proviral DNA integrated into the host genome. These latently infected cells (in lymph nodes, the gut, and the CNS) do not produce viral particles and are therefore not targeted by ART. If ART is stopped, the provirus can reactivate, leading to viral rebound.

(c) "Undetectable" means the viral load is below the assay's detection limit ( $< 50\;\mathrm{copies/mL}$ ), not that the virus is absent. Low levels of virus may still be present and potentially transmissible, though the risk is greatly reduced. The "U = U" (undetectable = untransmittable) consensus holds that individuals with sustained undetectable viral load have effectively zero risk of sexual transmission, but this requires consistent adherence to ART.

Question 3: Monoclonal Antibody Production

Describe the steps involved in producing monoclonal antibodies using the hybridoma technique. Explain why HAT medium is essential and why unfused B cells and unfused myeloma cells cannot survive in this medium. Calculate the number of hybridoma clones that must be screened if a researcher needs to identify one specific clone from a population of $50000$ fused cells, assuming $0.1\%$ of hybridomas produce the desired antibody.

Answer

Steps:

A mouse is immunised with the target antigen, stimulating B cell proliferation.
B cells are extracted from the mouse spleen.
B cells are fused with myeloma cells using polyethylene glycol (PEG).
The mixture is cultured in HAT medium.

HAT medium selection:

HAT medium contains hypoxanthine, aminopterin, and thymidine. Aminopterin blocks the de novo nucleotide synthesis pathway. Cells must use the salvage pathway (hypoxanthine-guanine phosphoribosyltransferase, HGPRT, and thymidine kinase, TK) to synthesise nucleotides.
Unfused myeloma cells: lack HGPRT (they are HGPRT $^-$ mutants selected for this purpose), so they cannot use the salvage pathway. With the de novo pathway blocked by aminopterin, they die.
Unfused B cells: have HGPRT but are short-lived (survive only $7$ -- $10$ days in culture) and die naturally.
Hybridomas: inherit HGPRT from the B cell and immortality from the myeloma cell. They survive and proliferate in HAT medium.

Screening calculation: Number of hybridomas producing the desired antibody: $50000 \times 0.001 = 50$ clones. The researcher must screen up to $50000$ clones (worst case) or, using a sampling strategy, could screen enough to find at least one positive clone. In practice, screening is done using ELISA on supernatant from each well of a multi-well plate.

Question 4: Transplant Rejection

A kidney transplant patient receives a kidney from a donor with HLA types A3, A11, B7, B35, DR2, DR4. The recipient has HLA types A1, A3, B8, B35, DR1, DR4. (a) How many HLA loci match? (b) Would you expect the risk of rejection to be high or low? Justify your answer. (c) The recipient is given cyclosporin A. Explain the mechanism of action of this drug and the specific immune process it suppresses.

Answer

(a) Matching HLA loci:

HLA-A: A3 matches (1 match); A11 vs A1 = mismatch.
HLA-B: B35 matches (1 match); B7 vs B8 = mismatch.
HLA-DR: DR4 matches (1 match); DR2 vs DR1 = mismatch.
Total: $3$ out of $6$ loci match ( $50\%$ match).

(b) A $50\%$ HLA match represents a moderate risk of rejection. Better-matched kidneys (higher number of shared HLA alleles) have lower rejection rates and longer graft survival. With $3$ mismatches, the immune system will recognise the foreign HLA antigens on the donor kidney, potentially triggering both T cell-mediated (acute) and antibody-mediated (chronic) rejection.

(c) Cyclosporin A binds to cyclophilin (an immunophilin) in the cytoplasm of T cells. The cyclosporin-cyclophilin complex inhibits calcineurin, a phosphatase that normally activates the transcription factor NFAT (nuclear factor of activated T cells). Without NFAT activation, transcription of IL-2 and other cytokine genes is blocked. Since IL-2 is essential for T cell clonal expansion and differentiation, cyclosporin A suppresses T cell-mediated immune responses against the transplanted organ.

Question 5: Allergic Response and Anaphylaxis

A student with a severe peanut allergy accidentally consumes a cookie containing peanut flour. Within minutes, they develop urticaria, facial swelling, wheezing, and a drop in blood pressure. (a) Describe the immunological mechanism responsible for these symptoms, naming the cells, antibodies, and chemical mediators involved. (b) Explain why the onset is rapid (minutes) rather than delayed. (c) Explain why adrenaline is the first-line treatment, relating its pharmacological effects to each symptom.

Answer

(a) This is a Type I hypersensitivity reaction:

The student was previously sensitised: peanut allergens stimulated $\mathrm{T_H}2$ cells to release IL-4, causing B cells to produce IgE anti-peanut antibodies.
IgE bound to $\mathrm{Fc\varepsilon}$ receptors on mast cells in connective tissue and basophils in blood.
Upon re-exposure, peanut allergens cross-linked IgE on sensitised mast cells.
Mast cells degranulated, releasing histamine (vasodilation, increased permeability, bronchoconstriction), leukotrienes (bronchoconstriction, increased vascular permeability), and prostaglandin $\mathrm{D}_2$ .

(b) The rapid onset (minutes) occurs because the sensitised mast cells already have allergen-specific IgE bound to their surface. Cross-linking of IgE triggers immediate degranulation of pre-formed mediators (histamine is stored in granules). There is no need for clonal expansion or antibody production --- the effector response is pre-armed.

Bronchoconstriction and wheezing: adrenaline is a $\beta_2$ -adrenergic agonist, causing relaxation of bronchial smooth muscle.
Vasodilation and hypotension: adrenaline activates $\alpha_1$ receptors on blood vessels, causing vasoconstriction that increases blood pressure.
Increased capillary permeability and swelling: vasoconstriction reduces capillary leakage.
Further mast cell degranulation: adrenaline activates $\beta_2$ receptors on mast cells, inhibiting further mediator release.

Worked Examples

Worked Example: Primary vs Secondary Immune Response Kinetics

A patient receives a tetanus toxoid vaccine (primary immunisation) at Day 0 and a booster dose at Day $90$ . The IgG antibody titres are measured weekly. At Day $14$ , the IgG titre is $50\;\mathrm{AU/mL}$ . At Day $21$ (after booster), the IgG titre is $2000\;\mathrm{AU/mL}$ . Calculate the fold increase in peak titre and the time to peak after the booster. Explain the cellular basis for the accelerated secondary response.

Solution

Fold increase: $\frac{2000}{50} = 40$ -fold increase in peak IgG titre.

Time to peak: the primary response peaked at approximately Day $14$ ( $14$ days post-immunisation). The secondary response peaked at approximately Day $7$ after the booster (Day $97$ total), so the time to peak decreased from $14$ days to approximately $7$ days.

Cellular basis:

During the primary response, naive B cells (frequency $\approx 1$ in $10^5$ ) must be activated, undergo clonal expansion ( $3$ -- $5$ days), differentiate into plasma cells, and begin secreting IgM followed by class-switched IgG. Memory B cells and memory T cells are generated.
During the secondary response, memory B cells (present at $\approx 100$ -fold higher frequency than the original naive clone) are rapidly reactivated. Memory B cells have already undergone class switching (to IgG) and affinity maturation (higher-affinity BCRs). They differentiate into plasma cells within $1$ -- $3$ days, producing high-affinity IgG immediately. Memory helper T cells provide rapid cytokine support.
The secondary response is faster, stronger, and produces higher-affinity antibodies, providing effective protection against the pathogen.

Worked Example: Interpreting a Mantoux Test

A Mantoux test is performed by injecting $5$ tuberculin units ( $0.1\;\mathrm{mL}$ ) of purified protein derivative (PPD) intradermally. The induration (raised, hardened area) is measured $48$ -- $72$ hours later. Three patients show the following results: Patient X = $5\;\mathrm{mm}$ , Patient Y = $12\;\mathrm{mm}$ , Patient Z = $18\;\mathrm{mm}$ . Patient Y is known to be immunocompromised. Interpret each result.

Solution

Interpretation guidelines (based on CDC criteria):

Patient X ( $5\;\mathrm{mm}$ ): Negative. An induration of $< 5\;\mathrm{mm}$ is considered negative regardless of risk factors. No evidence of M. tuberculosis infection.
Patient Y ( $12\;\mathrm{mm}$ ): Positive. For immunocompromised individuals, an induration $\geq 5\;\mathrm{mm}$ is considered positive. The patient has likely been infected with M. tuberculosis (latent TB infection). Further investigation (chest X-ray, sputum culture) is needed.
Patient Z ( $18\;\mathrm{mm}$ ): Positive. An induration $\geq 10\;\mathrm{mm}$ is positive for most individuals (healthcare workers, recent immigrants, IV drug users). An induration $\geq 15\;\mathrm{mm}$ is positive for all individuals regardless of risk factors.

Immunological basis: the Mantoux test is a Type IV (delayed-type) hypersensitivity reaction. Memory $\mathrm{T_H}1$ cells recognise PPD antigens and release IFN- $\gamma$ , activating macrophages. Macrophages release inflammatory mediators, causing localised oedema and induration. The $48$ -- $72$ hour delay reflects the time required for T cell activation, cytokine secretion, and macrophage recruitment.

Common Pitfalls (Expanded)

Confusing antigens and antibodies: antigens are molecules that provoke an immune response; antibodies are the proteins produced in response. Antigens are on the pathogen surface; antibodies are produced by plasma cells.
Stating that B cells directly kill infected cells: B cells secrete antibodies (humoral immunity); cytotoxic T cells and NK cells directly kill infected cells (cell-mediated immunity).
Confusing IgG and IgM in primary vs secondary responses: IgM is produced first in the primary response; IgG dominates the secondary response due to class switching and memory B cell activation.
Describing HIV as killing all white blood cells: HIV specifically targets $\mathrm{CD4}^+$ helper T cells. The resulting immunodeficiency is indirect, caused by the loss of T cell help to B cells and cytotoxic T cells.
Confusing autoimmunity and immunodeficiency: autoimmunity is immune overactivity against self; immunodeficiency is immune underactivity or absence.
Assuming all allergies are IgE-mediated: Type I hypersensitivity (allergy, asthma, anaphylaxis) is IgE-mediated, but other hypersensitivity types exist: Type II (antibody-mediated cytotoxicity), Type III (immune complex disease), Type IV (delayed-type, T-cell mediated).
Stating that monoclonal antibodies come from a single B cell: they come from a single hybridoma (B cell fused with myeloma cell). A normal B cell cannot proliferate indefinitely.
Confusing the classical and alternative complement pathways: the classical pathway requires antibody-antigen complexes; the alternative pathway is activated spontaneously on microbial surfaces and is part of innate immunity.

Exam-Style Problems

Problem 1: Extended Response -- Comparing Innate and Adaptive Immunity

Compare and contrast innate and adaptive immunity with reference to: (a) specificity, (b) response time, (c) memory, (d) receptors, and (e) the role of phagocytes in each. Explain how innate immunity activates adaptive immunity, describing the process of antigen presentation by dendritic cells to helper T cells.

Problem 2: Data Analysis -- Antibody Titre Curve

The following data show the IgM and IgG antibody concentrations (in arbitrary units, AU/mL) in a patient's serum after vaccination at Day 0 and a booster at Day 90:

Day post-vaccination	IgM (AU/mL)	IgG (AU/mL)
0	0	0
5	20	5
10	80	40
14	120	100
21	60	150
28	30	120
60	10	80
90 (booster)	15	90
93	25	800
97	30	2000
104	20	1500
120	10	1000

(a) Plot both IgM and IgG curves on the same axes. (b) Identify the class switching event and explain its molecular basis. (c) Calculate the fold increase in peak IgG titre between the primary and secondary responses. (d) Explain why IgM peaks earlier than IgG in the primary response but not in the secondary response.

Problem 3: Extended Response -- HIV Life Cycle and Drug Targets

Describe the HIV life cycle from attachment to budding, naming the viral and host molecules involved at each step. For each of the following drug classes, identify the step of the life cycle they target and explain their mechanism: (a) NRTIs, (b) protease inhibitors, (c) CCR5 antagonists, (d) integrase inhibitors. Explain why combination therapy (using drugs from multiple classes) is more effective than monotherapy, and discuss why HIV can develop resistance to individual drugs.

Problem 4: Extended Response -- ELISA Design

A medical diagnostic company wants to develop an ELISA to detect a new viral antigen in patient blood samples. (a) Should they design a direct, indirect, or sandwich ELISA? Justify your choice. (b) Describe each step of the chosen ELISA protocol, including the necessary controls. (c) Explain how the sensitivity and specificity of the test could be affected by cross-reactive antibodies in the patient sample. (d) Calculate the minimum detectable concentration of antigen if the assay produces a detectable colour change at an absorbance of $0.100$ and the standard curve has a slope of $0.020\;\mathrm{(AU/mL)}^{-1}$ with a y-intercept of $0.050$ .

Problem 5: Extended Response -- Autoimmunity and Molecular Mimicry

Rheumatic fever is an autoimmune disease that can develop after infection with Streptococcus pyogenes (Group A Streptococcus). (a) Explain the concept of molecular mimicry and how it could lead to rheumatic fever. (b) Describe the immune mechanisms that damage the heart valves in rheumatic fever, including the specific antibodies and T cells involved. (c) Explain why prompt antibiotic treatment of streptococcal pharyngitis reduces the risk of developing rheumatic fever.

If You Get These Wrong, Revise:

Cell biology and phagocytosis --> Review ./cell-biology
Molecular biology and protein structure --> Review ./molecular-biology
Genetics and V(D)J recombination --> Review ./genetics
Human physiology -- circulatory system --> Review ./human-physiology
Enzyme kinetics (ELISA) --> Review ./metabolism-cell-biology

Additional Worked Examples

Worked Example: Calculating Antibody Titre

A patient's serum is serially diluted and tested for the presence of IgM antibodies against a pathogen:

Dilution	Antibody detected?
1:2	Yes
1:4	Yes
1:8	Yes
1:16	Yes
1:32	Yes
1:64	Yes
1:128	No
1:256	No

(a) What is the antibody titre? (b) A convalescent sample from the same patient taken 2 weeks later shows a titre of $1:2048$ . Calculate the fold increase. (c) Explain the clinical significance of a rising titre. (d) Why is IgM detection preferred for early diagnosis of acute infection?

Solution

(a) The titre is the highest dilution at which the antibody is still detectable: $1:64$ .

(b) Fold increase $= 2048 / 64 = 32$ -fold increase.

(c) A rising titre (4-fold or greater increase between acute and convalescent samples) indicates a recent or ongoing infection. It demonstrates that the immune system is actively producing antibodies against the pathogen, confirming acute rather than past infection or prior vaccination. A single positive result cannot distinguish between past and current infection; the rising titre provides this temporal information.

(d) IgM is the first antibody class produced in a primary immune response (appears within $1$ -- $2$ weeks of infection, peaks at $2$ -- $4$ weeks, then declines). IgG appears later ( $2$ -- $4$ weeks) and persists long-term. Detection of IgM therefore indicates a recent or current infection, while IgG indicates either past infection, vaccination, or a later stage of current infection. IgM is also a pentamer (10 antigen-binding sites), making it very effective at agglutination and complement activation even at low concentrations.

Worked Example: Clonal Selection and Memory Cell Calculation

In a primary immune response, a single B cell with the appropriate receptor is activated and expands into a clone of $5000$ plasma cells and $200$ memory B cells. Each plasma cell secretes $1000$ antibody molecules per second. (a) Calculate the total antibody production rate of the clone. (b) Upon secondary exposure, memory B cells rapidly expand. If each memory B cell gives rise to a clone of $10\,000$ plasma cells, calculate the secondary response antibody production rate. (c) Calculate the fold increase in antibody production rate between primary and secondary responses. (d) Explain why the secondary response is faster and produces more antibody.

Solution

(a) Primary response: $5000$ plasma cells $\times 1000$ antibodies/s $= 5 \times 10^6$ antibodies/s from this clone. In practice, multiple B cell clones are activated (polyclonal response), but this illustrates the principle for a single clone.

(b) Secondary response: $200$ memory B cells $\times 10\,000$ plasma cells each $= 2 \times 10^6$ plasma cells. Antibody production rate: $2 \times 10^6 \times 1000 = 2 \times 10^9$ antibodies/s.

(d) The secondary response is faster and stronger because:

More memory cells: the primary response generates many memory B cells ( $200$ in this example) that persist for years. Upon re-exposure, all memory cells are activated simultaneously.
Higher affinity: memory B cells have undergone somatic hypermutation and affinity maturation in germinal centres during the primary response. Their receptors have higher affinity for the antigen, leading to more efficient activation and antibody production.
Class switching: memory B cells have already undergone class switching (to IgG, IgA, or IgE), allowing immediate production of high-affinity IgG (which is more effective than IgM).
Isotype switching to IgG: IgG has a longer half-life ( $\approx 21$ days vs $5$ days for IgM), better opsonisation (binds Fc receptors on phagocytes), and activates complement more efficiently.
Faster kinetics: memory B cells are pre-activated (express lower activation thresholds) and require less co-stimulation than naive B cells.

Worked Example: ELISA Calculation

A sandwich ELISA is used to measure the concentration of a viral antigen in patient serum. The standard curve is generated using known antigen concentrations:

Antigen concentration (ng/mL)	Absorbance at 450 nm
0	0.050
5	0.150
10	0.250
20	0.450
40	0.850
80	1.650

A patient sample gives an absorbance of $0.550$ . (a) Plot the standard curve and determine the antigen concentration in the patient sample. (b) The positive threshold (determined from negative controls) is $0.200$ . Is the patient positive? (c) Calculate the signal-to-noise ratio if the negative control absorbance is $0.060$ . (d) Explain the principle of the sandwich ELISA and why two antibodies are required.

Solution

(a) Using the standard curve (approximately linear from $0$ to $40\;\mathrm{ng/mL}$ ): Slope $= (0.850 - 0.050) / (40 - 0) = 0.800 / 40 = 0.020\;\mathrm{(AU \cdot mL)/ng}$ . Y-intercept $= 0.050$ .

For the patient sample: $0.550 = 0.020 \times C + 0.050$ . $C = (0.550 - 0.050) / 0.020 = 0.500 / 0.020 = 25\;\mathrm{ng/mL}$ .

(b) Patient absorbance $= 0.550 > 0.200$ (positive threshold). The patient is positive for the viral antigen.

(c) Signal-to-noise ratio $= \text{patient signal} / \text{negative control} = 0.550 / 0.060 = 9.2$ . A ratio $> 3$ is generally considered significant; this result is well above that threshold.

(d) Sandwich ELISA principle:

A capture antibody (specific to the antigen) is coated on the bottom of the microplate well.
Patient sample is added. If the antigen is present, it binds to the capture antibody.
A detection antibody (also specific to the antigen, but binding to a different epitope) is added. This antibody is conjugated to an enzyme (e.g., horseradish peroxidase, HRP).
A substrate for the enzyme is added. The enzyme converts the substrate to a coloured product.
The absorbance is proportional to the amount of antigen in the sample.

Two antibodies are needed because: (1) the capture antibody immobilises the antigen; (2) the detection antibody provides specificity and signal amplification. Using two antibodies that bind different epitopes increases assay specificity (reducing false positives from cross-reactive substances).

Worked Example: HIV and the Immune System

HIV targets $\mathrm{CD4}^+$ T helper cells. A patient's blood test shows: $\mathrm{CD4}^+$ T cell count $= 350\;\mathrm{cells/\mu L}$ (normal: $500$ -- $1600$ ), viral load $= 50\,000\;\mathrm{copies/mL}$ . (a) What stage of HIV infection does this represent? (b) Explain how HIV depletes $\mathrm{CD4}^+$ cells. (c) Explain why opportunistic infections occur at this stage. (d) Explain how antiretroviral therapy (ART) works.

Solution

(a) $\mathrm{CD4}^+$ count of $350\;\mathrm{cells/\mu L}$ falls in the range of $200$ -- $500$ , which corresponds to stage 2 (chronic HIV infection) or early stage 3. AIDS is defined as $\mathrm{CD4}^+ < 200\;\mathrm{cells/\mu L}$ or the presence of an AIDS-defining illness. This patient has not yet progressed to AIDS but has significant immunosuppression.

(b) HIV depletes $\mathrm{CD4}^+$ T cells through multiple mechanisms:

Direct viral killing: HIV replicates inside $\mathrm{CD4}^+$ cells, producing new virions that bud from the cell membrane, causing cell lysis.
Syncytium formation: HIV envelope glycoproteins on infected cells bind to $\mathrm{CD4}$ on uninfected cells, causing cell fusion and forming multinucleated syncytia that die.
Apoptosis: HIV infection triggers programmed cell death through various pathways (Fas/FasL, caspase activation).
Immune-mediated killing: cytotoxic T lymphocytes (CTLs) recognise and kill HIV-infected $\mathrm{CD4}^+$ cells (this is beneficial for controlling the virus but also depletes $\mathrm{CD4}^+$ cells).
Chronic immune activation: persistent viral replication keeps the immune system activated, leading to exhaustion and apoptosis of uninfected bystander $\mathrm{CD4}^+$ cells.

(c) Opportunistic infections occur because $\mathrm{CD4}^+$ T helper cells are essential for coordinating both cell-mediated and humoral immune responses:

They activate macrophages (via IFN- $\gamma$ ) to kill intracellular pathogens.
They provide help to B cells for antibody production (CD40L-CD40 interaction, cytokine secretion).
They activate CTLs (via IL-2).
With reduced $\mathrm{CD4}^+$ cells, all these functions are impaired. Pathogens that are normally controlled (e.g., Pneumocystis jirovecii, Mycobacterium avium, Candida, cytomegalovirus) cause severe, life-threatening infections.

(d) Antiretroviral therapy (ART) typically uses a combination of three drugs from at least two classes:

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): competitive substrates that cause chain termination during viral DNA synthesis (e.g., AZT/zidovudine, tenofovir).
Non-nucleoside reverse transcriptase inhibitors (NNRTIs): allosteric inhibitors that bind RT and change its conformation, preventing DNA synthesis (e.g., efavirenz).
Protease inhibitors (PIs): inhibit HIV protease, preventing cleavage of viral polyproteins into functional proteins (e.g., ritonavir).
Integrase inhibitors: block HIV integrase, preventing integration of viral DNA into the host genome (e.g., raltegravir).
Entry inhibitors/fusion inhibitors: block viral entry (e.g., maraviroc blocks CCR5 co-receptor; enfuvirtide blocks gp41-mediated fusion).

ART does not cure HIV (the latent reservoir persists) but suppresses viral replication to undetectable levels, allowing immune recovery and preventing disease progression.

Additional Common Pitfalls

Confusing antigens and antibodies: antigens are molecules that trigger an immune response; antibodies are proteins produced by B cells that bind to specific antigens.
Confusing active and passive immunity: active immunity = the body produces its own antibodies (natural infection or vaccination); passive immunity = pre-formed antibodies are transferred (maternal IgG across placenta, antivenom injection).
Assuming all bacteria are pathogens: most bacteria are harmless or beneficial; only a minority are pathogenic.
Confusing primary and secondary immune responses: primary response = slow, weak, IgM first; secondary response = fast, strong, predominantly IgG (memory-dependent).
Confusing T cells and B cells: T cells mediate cell-mediated immunity (CTLs kill infected cells; helper T cells coordinate the response); B cells produce antibodies (humoral immunity).
Assuming vaccines provide immediate protection: most vaccines require time (weeks) for the primary response and booster doses to develop full immunity. Some vaccines require multiple doses.

Additional Exam-Style Problems with Full Solutions

Problem 6: Extended Response -- Complement System

Describe the three pathways of complement activation (classical, lectin, alternative). For each pathway: (a) identify the trigger, (b) describe the key steps leading to C3 convertase formation, (c) explain the common terminal pathway (membrane attack complex, MAC), and (d) describe three biological consequences of complement activation. (e) Explain how complement regulation prevents damage to host cells.

Answer 6

Classical pathway: (a) Trigger: antibody-antigen complexes (IgM or IgG bound to antigen). (b) Steps: C1q binds to the Fc region of antibody. This activates C1r and C1s (serine proteases). C1s cleaves C4 into C4a and C4b, and C2 into C2a and C2b. C4b2a forms the C3 convertase on the pathogen surface.

Lectin pathway: (a) Trigger: mannose-binding lectin (MBL) binds to mannose residues on microbial surfaces. (b) Steps: MBL is associated with MASP-1 and MASP-2 (mannose-associated serine proteases), analogous to C1r/C1s. MASP-2 cleaves C4 and C2, forming the same C4b2a C3 convertase.

Alternative pathway: (a) Trigger: spontaneous hydrolysis of C3 (tickover) on microbial surfaces. Foreign surfaces lack regulatory proteins (e.g., factor H), allowing amplification. (b) Steps: C3b binds to the microbial surface and combines with factor B. Factor D cleaves factor B, forming C3bBb (the alternative pathway C3 convertase). This is stabilised by properdin (factor P).

Common terminal pathway (all three pathways): The C3 convertase cleaves C3 into C3a (anaphylatoxin) and C3b (opsonin). C3b binds to the surface and combines with the convertase to form C5 convertase. C5 convertase cleaves C5 into C5a (anaphylatoxin, chemotactic factor) and C5b. C5b initiates the assembly of the membrane attack complex (MAC): C5b recruits C6, C7, C8, and multiple C9 molecules. C9 polymerises to form a transmembrane pore (approximately $10\;\mathrm{nm}$ diameter) that disrupts the target cell's membrane integrity, causing osmotic lysis.

(d) Three biological consequences:

Opsonisation: C3b deposited on the pathogen surface binds to CR1 (complement receptor 1) on phagocytes, enhancing phagocytosis.
Inflammation: C3a and C5a (anaphylatoxins) cause mast cell degranulation (histamine release), vasodilation, increased vascular permeability, and recruitment of neutrophils (C5a is a potent chemoattractant).
Cell lysis: the MAC forms pores in the membrane of gram-negative bacteria, enveloped viruses, and parasite-infected cells, causing osmotic lysis and death.

(e) Complement regulation prevents damage to host cells:

Decay-accelerating factor (DAF, CD55): dissociates C3 convertases on host cell surfaces.
Membrane cofactor protein (MCP, CD46): acts as a cofactor for factor I-mediated cleavage of C3b and C4b on host cells.
Factor H: soluble regulator that binds to host cell surfaces (via sialic acid and glycosaminoglycans) and promotes C3b inactivation by factor I.
CD59 (protectin): inhibits MAC formation by preventing C9 polymerisation on host cells.
C1 inhibitor (C1-INH): inactivates C1r and C1s, preventing excessive classical pathway activation.
Viral evasion: some viruses produce complement regulatory protein homologues (e.g., vaccinia virus VCP, herpesvirus complement control proteins) to evade complement-mediated destruction.

Problem 7: Data Analysis -- Blood Type Genetics and Paternity

A mother has blood type A (genotype unknown) and a child has blood type O. (a) What are the possible genotypes of the mother? (b) What blood types are possible for the father? (c) If a man with blood type AB is accused of being the father, can he be excluded? (d) Explain why blood type alone is insufficient for definitive paternity testing.

Answer 7

(a) The mother is blood type A. Possible genotypes: $I^A I^A$ or $I^A i$ .

(b) The child is blood type O (genotype $ii$ ). Each parent must contribute an $i$ allele. Therefore, the mother must be $I^A i$ (she contributed the $i$ allele). The father must also contribute an $i$ allele, so the father must have at least one $i$ allele. Possible father genotypes: $I^A i$ (blood type A), $I^B i$ (blood type B), or $ii$ (blood type O).

(c) The accused man has blood type AB (genotype $I^A I^B$ ). He can only contribute $I^A$ or $I^B$ alleles to his offspring. He cannot contribute an $i$ allele. Therefore, he cannot be the father of a blood type O child. He is excluded.

(d) Blood type alone is insufficient for definitive paternity testing because:

Many men share the same blood type (e.g., approximately $36\%$ of the population is blood type O; a very large number of men cannot be excluded based on blood type O alone).
Blood typing can only exclude a man, never definitively include him (it provides exclusion, not identification).
Modern paternity testing uses DNA profiling (STR analysis), which examines multiple highly variable genetic loci and can provide paternity probabilities $> 99.99\%$ .

Problem 8: Extended Response -- Monoclonal Antibodies in Medicine

Describe the production of monoclonal antibodies using the hybridoma technique (Kohler and Milstein, 1975). (a) Explain each step of the process. (b) Explain why myeloma cells and B cells are fused. (c) Describe three therapeutic applications of monoclonal antibodies, explaining the mechanism of action for each. (d) Explain the difference between murine, chimeric, humanised, and fully human monoclonal antibodies.

Answer 8

(a) Hybridoma technique:

Immunisation: a mouse is injected with the target antigen. The mouse's immune system produces B cells that secrete antibodies specific to the antigen.
Cell harvest: splenocytes (including antibody-producing B cells) are harvested from the mouse's spleen.
Cell fusion: splenocytes are fused with myeloma cells (immortal cancer cells) using polyethylene glycol (PEG) or electrofusion.
Selection: the cell mixture is cultured in HAT medium (hypoxanthine-aminopterin-thymidine). Myeloma cells lack HGPRT (an enzyme for the salvage pathway) and die. Unfused B cells have limited lifespan and die. Only hybridomas (fused B cell + myeloma) survive because the B cell provides HGPRT and the myeloma provides immortality.
Screening: hybridoma supernatants are tested for the desired antibody (using ELISA or similar).
Cloning: positive hybridomas are subcloned by limiting dilution (single cell per well) to ensure monoclonality.
Expansion and storage: the monoclonal hybridoma is expanded in culture or in mice (ascites production). Cells are cryopreserved.

(b) Why fuse myeloma and B cells: B cells produce the desired antibody but cannot divide indefinitely in culture (they die after a few divisions). Myeloma cells are immortal (divide indefinitely) but do not produce the desired antibody. The hybridoma combines the antibody-producing capability of the B cell with the immortality of the myeloma cell.

Trastuzumab (Herceptin): a monoclonal antibody against HER2 (human epidermal growth factor receptor 2), overexpressed in approximately $25\%$ of breast cancers. Mechanism: binds HER2, blocking downstream signalling (inhibiting cell proliferation), and flags the cancer cell for destruction by NK cells (ADCC -- antibody-dependent cellular cytotoxicity).
Rituximab: targets CD20 on B cells. Used in non-Hodgkin lymphoma and rheumatoid arthritis. Mechanism: binds CD20, causing complement-dependent cytotoxicity (CDC), ADCC, and direct apoptosis of B cells. This depletes malignant or auto-reactive B cells.
Adalimumab (Humira): a monoclonal antibody against TNF- $\alpha$ (tumour necrosis factor-alpha). Used in rheumatoid arthritis, Crohn's disease, and other autoimmune conditions. Mechanism: binds and neutralises TNF- $\alpha$ , preventing it from binding to its receptors and triggering inflammation.

(d) Antibody types by human content:

Murine: $100\%$ mouse protein. High immunogenicity in humans (human anti-mouse antibody, HAMA, response). Largely replaced by newer types.
Chimeric: approximately $65\%$ human (constant regions), $35\%$ mouse (variable regions). Named with "-ximab" suffix (e.g., rituximab). Reduced immunogenicity compared to murine.
Humanised: approximately $90\%$ human (all but the CDRs, complementarity-determining regions, are human). Named with "-zumab" suffix (e.g., trastuzumab). Lower immunogenicity.
Fully human: $100\%$ human protein. Produced using phage display or transgenic mice with human immunoglobulin genes. Named with "-umab" suffix (e.g., adalimumab). Minimal immunogenicity.

Problem 9: Extended Response -- Hypersensitivity Reactions

Compare and contrast the four types of hypersensitivity reactions (I, II, III, IV). For each type: (a) give the mechanism, (b) provide a specific example, (c) state the time course, and (d) identify the key effector molecules or cells involved.

Answer 9

Feature	Type I (Immediate)	Type II (Cytotoxic)	Type III (Immune complex)	Type IV (Delayed-type)
Mechanism	IgE-mediated; mast cell degranulation	IgG/IgM against cell-surface antigens; complement activation, ADCC	Immune complexes deposit in tissues; complement activation, neutrophil recruitment	T cell-mediated (Th1 and CTL); delayed hypersensitivity
Time course	Minutes (immediate); late phase 2--8 hours	Hours to days	Hours to days (serum sickness: 7--10 days)	48--72 hours (delayed)
Example	Asthma, hay fever, anaphylaxis, food allergy	Goodpasture syndrome, transfusion reactions, haemolytic disease of newborn	Serum sickness, systemic lupus erythematosus (SLE), Arthus reaction	Tuberculin skin test (Mantoux), contact dermatitis (poison ivy), type 1 diabetes
Key effectors	IgE, mast cells, basophils, eosinophils, histamine, leukotrienes	IgG, IgM, complement (MAC), NK cells (ADCC), macrophages	IgG, IgM, complement (C3a, C5a), neutrophils, MAC	Th1 cells (IFN- $\gamma$ , IL-2), CTLs (perforin, granzyme), macrophages
Antibody involved?	Yes (IgE)	Yes (IgG, IgM)	Yes (IgG, IgM)	No (T cell-mediated)

(d) Type I is mediated by IgE binding to Fc receptors on mast cells, triggering degranulation (histamine, heparin, tryptase, leukotrienes). Type II involves antibodies targeting specific cells (e.g., blood cells). Type III involves circulating immune complexes that deposit in small blood vessels, joints, and kidneys, triggering inflammation. Type IV is the only type not involving antibodies; it is mediated by sensitised T cells.

Cell membrane and receptor proteins: Review ./cell-biology for membrane protein structure and signalling.
Protein structure and antibodies: Review ./molecular-biology for immunoglobulin structure and antigen binding.
Genetics and V(D)J recombination: Review ./genetics for antibody diversity generation.
Human physiology -- blood and circulation: Review ./human-physiology for blood cell types and circulatory pathways.
Molecular biology -- DNA technology: Review ./genetics-advanced for monoclonal antibody production techniques.

Supplementary: Antibody Diversity -- V(D)J Recombination (HL Extension)

The Challenge of Antibody Diversity

The adaptive immune system must be able to recognise an essentially unlimited number of different antigens (estimated $> 10^{11}$ different specificities). However, the human genome contains only approximately $20\,000$ protein-coding genes. The solution is the generation of antibody diversity through combinatorial and somatic mechanisms during B cell development in the bone marrow.

Antibody Structure Recap

An immunoglobulin (Ig) molecule consists of:

Two identical heavy chains ( $\mu$ , $\delta$ , $\gamma$ , $\epsilon$ , or $\alpha$ -- determining the antibody class: IgM, IgD, IgG, IgE, or IgA).
Two identical light chains ( $\kappa$ or $\lambda$ ).

Each chain has:

Variable region ( $V$ ): at the N-terminus, contains the antigen-binding site. The heavy chain variable region ( $V_H$ ) and light chain variable region ( $V_L$ ) together form the antigen-binding site (two per antibody molecule).
Constant region ( $C$ ): determines the effector function (complement activation, Fc receptor binding, placental transfer).

V(D)J Recombination

The variable region of the heavy chain is encoded by three gene segments: V (variable), D (diversity), and J (joining). The light chain uses only V and J segments.

Heavy chain locus (chromosome 14):

Approximately $40$ -- $45$ functional V gene segments
$23$ functional D gene segments
$6$ functional J gene segments

Light chain loci:

$\kappa$ locus (chromosome 2): approximately $40$ V, $5$ J segments
$\lambda$ locus (chromosome 22): approximately $30$ V, $4$ J segments

During B cell development:

A D segment randomly joins to a J segment (D-J joining).
A V segment randomly joins to the D-J unit (V-DJ joining).
This forms a complete VDJ unit, which is transcribed together with the $\mu$ constant region to produce the heavy chain of IgM.
The light chain undergoes V-J joining (either $\kappa$ or $\lambda$ locus) to produce the light chain.
Any successful heavy-light chain combination produces a pre-B cell receptor, followed by a B cell receptor.

Sources of Antibody Diversity

Combinatorial diversity: the random combination of V, D, and J segments produces many different variable regions. Heavy chain: $45 \times 23 \times 6 = 6210$ combinations. Light chain: approximately $40 \times 5 = 200$ ( $\kappa$ ) or $30 \times 4 = 120$ ( $\lambda$ ). Combined: $6210 \times 320 \approx 2 \times 10^6$ combinations.
Junctional diversity: during V-D and D-J joining, the enzyme terminal deoxynucleotidyl transferase (TdT) adds random nucleotides (P nucleotides and N nucleotides) at the junctions. Additionally, exonucleases may trim nucleotides from the ends of the segments before joining. This imprecise joining dramatically increases diversity, contributing an estimated $3 \times 10^7$ additional combinations.
Combinatorial association: any heavy chain can pair with any light chain, further multiplying diversity.
Somatic hypermutation: after antigen exposure, activated B cells in germinal centres undergo point mutations in the variable region at a rate of approximately $10^{-3}$ per base pair per generation ( $10^6\times$ the normal mutation rate). This produces B cell clones with higher or lower affinity for the antigen. Those with higher affinity are selected for survival (affinity maturation).
Class switching: the constant region of the heavy chain can be changed (from IgM to IgG, IgA, or IgE) by a DNA recombination event called switch recombination. The variable region (and therefore antigen specificity) remains the same, but the effector function changes.

Worked Example: Calculating Antibody Diversity

Given the following numbers of functional gene segments:

Heavy chain: $44$ V, $27$ D, $6$ J
$\kappa$ light chain: $38$ V, $5$ J
$\lambda$ light chain: $29$ V, $4$ J

(a) Calculate the combinatorial diversity from V(D)J recombination alone. (b) If junctional diversity adds a factor of approximately $10^7$ for the heavy chain, calculate the total diversity including junctional and combinatorial mechanisms. (c) If somatic hypermutation introduces an average of $5$ mutations per variable region (which has $300\;\mathrm{bp}$ ), and each mutation can be one of $3$ possible nucleotide changes, calculate the additional diversity from somatic hypermutation for a single B cell clone.

Solution

(a) Heavy chain combinations: $44 \times 27 \times 6 = 7128$ . $\kappa$ light chain: $38 \times 5 = 190$ . $\lambda$ light chain: $29 \times 4 = 116$ . Total light chain: $190 + 116 = 306$ . Combinatorial diversity: $7128 \times 306 = 2\,181\,168 \approx 2.2 \times 10^6$ .

(b) With junctional diversity ( $\times 10^7$ for heavy chain): $7128 \times 10^7 \times 306 = 2.2 \times 10^{13}$ .

(c) Somatic hypermutation: for $5$ mutations in $300\;\mathrm{bp}$ , each mutation has $3$ possible nucleotide changes. The number of distinct sequences generated: $\binom{300}{5} \times 3^5 = \frac{300!}{5! \times 295!} \times 243$ $\binom{300}{5} \approx 2.05 \times 10^{10}$ $2.05 \times 10^{10} \times 243 \approx 5 \times 10^{12}$

So a single B cell clone can generate approximately $5 \times 10^{12}$ variant sequences through somatic hypermutation. In practice, not all variants will be viable (some will introduce stop codons or destabilise the protein), but this illustrates the enormous potential for affinity maturation.

The Major Histocompatibility Complex (MHC)

The MHC (called HLA in humans -- Human Leukocyte Antigen) is a cluster of genes on chromosome 6 that encode cell-surface proteins responsible for presenting antigens to T cells.

Class I MHC (HLA-A, -B, -C):

Expressed on all nucleated cells.
Present endogenous antigens (intracellular proteins, e.g., viral proteins, tumour antigens) to $\mathrm{CD8}^+$ cytotoxic T cells (CTLs).
Structure: heavy chain ( $\alpha$ chain) + $\beta_2$ -microglobulin. The antigen-binding groove is formed by the $\alpha_1$ and $\alpha_2$ domains.
Pathway: intracellular proteins are degraded by the proteasome, transported into the ER by TAP (transporter associated with antigen processing), loaded onto MHC class I, and displayed on the cell surface.

Class II MHC (HLA-DR, -DP, -DQ):

Expressed on antigen-presenting cells (dendritic cells, macrophages, B cells).
Present exogenous antigens (extracellular proteins, e.g., bacteria, toxins) to $\mathrm{CD4}^+$ helper T cells.
Structure: $\alpha$ chain + $\beta$ chain. The antigen-binding groove is formed by the $\alpha_1$ and $\beta_1$ domains.
Pathway: extracellular proteins are endocytosed, degraded in endolysosomes, loaded onto MHC class II (assisted by HLA-DM), and displayed on the cell surface.

Polymorphism: MHC genes are the most polymorphic in the human genome (thousands of alleles per gene). This diversity ensures that at least some individuals in a population can present peptides from any pathogen (balancing selection). MHC differences between individuals are the molecular basis for transplant rejection (the immune system recognises foreign MHC as non-self).

T Cell Development and Selection

T cells develop in the thymus from bone marrow-derived progenitors:

Double-negative stage ( $\mathrm{CD4}^-\mathrm{CD8}^-$ ): T cell receptor (TCR) beta chain rearrangement occurs. Successful rearrangement produces the pre-TCR, which signals for proliferation and alpha chain rearrangement.
Double-positive stage ( $\mathrm{CD4}^+\mathrm{CD8}^+$ ): TCR alpha chain rearrangement completes. Cells that produce a functional TCR are selected for further development.
Positive selection (in the thymic cortex): double-positive T cells interact with cortical thymic epithelial cells expressing MHC class I and class II. T cells whose TCR can recognise self-MHC (weakly) receive survival signals. T cells that cannot recognise any self-MHC die by neglect (apoptosis). This ensures MHC restriction (T cells only recognise antigens presented by self-MHC).
Negative selection (in the thymic medulla): T cells that react strongly to self-antigens presented by self-MHC are deleted by apoptosis (clonal deletion). This eliminates self-reactive T cells and establishes central tolerance. Medullary thymic epithelial cells (mTECs) express AIRE (autoimmune regulator), which promotes the expression of tissue-specific antigens in the thymus, allowing negative selection against these antigens.
Single-positive stage: surviving T cells become either $\mathrm{CD4}^+$ (if selected on MHC class II) or $\mathrm{CD8}^+$ (if selected on MHC class I) and exit the thymus as naive T cells.

Approximately $98\%$ of developing T cells die in the thymus (negative selection and failed positive selection). Only about $2\%$ survive to enter the circulation as mature, self-tolerant T cells.

Types of Hypersensitivity -- Extended Table

Feature	Type I (Immediate)	Type II (Cytotoxic)	Type III (Immune complex)	Type IV (Delayed)
Onset	Seconds--minutes	Hours--days	Hours--days	48--72 hours
Mediator	IgE, mast cells	IgG, IgM, complement	IgG, IgM, complement	T cells (Th1, CTL)
Mechanism	Allergen cross-links IgE on mast cells	Antibody binds cell surface antigen	Circulating immune complexes deposit in tissues	Sensitised T cells release cytokines
Example	Hay fever, anaphylaxis	Haemolytic disease of newborn	Serum sickness	Contact dermatitis, TB test
Treatment	Antihistamines, adrenaline, allergen avoidance	Immunosuppressants, plasmapheresis	Anti-inflammatory drugs, immunosuppressants	Topical corticosteroids, calcineurin inhibitors

ABO Blood Group System -- Extended Details

The ABO blood group is determined by the presence of specific carbohydrate antigens on the surface of red blood cells:

Type A: N-acetylgalactosamine (GalNAc) antigen. Anti-B antibodies in plasma.
Type B: Galactose antigen. Anti-A antibodies in plasma.
Type AB: both A and B antigens. No anti-A or anti-B antibodies.
Type O: neither A nor B antigen. Both anti-A and anti-B antibodies.

The antigens are synthesised by glycosyltransferases encoded by the ABO gene on chromosome 9. The O allele encodes an inactive enzyme (frameshift mutation), so no antigen is produced.

Universal donor: type O (no A or B antigens to trigger recipient's antibodies). Can donate red blood cells to any type in emergencies (but only after cross-matching for other antigens).

Universal recipient: type AB (no anti-A or anti-B antibodies to react with donor antigens). Can receive red blood cells from any type.

Haemolytic disease of the newborn (HDNB): if an Rh-negative mother carries an Rh-positive fetus, she may produce anti-Rh (anti-D) IgG antibodies (usually during the second pregnancy, when fetal red cells cross the placenta during delivery and sensitise the mother). These IgG antibodies cross the placenta in subsequent pregnancies and destroy fetal Rh-positive red blood cells, causing anaemia, jaundice, oedema, and in severe cases, hydrops fetalis (heart failure). Prevention: RhoGAM (anti-D immunoglobulin) is administered to the Rh-negative mother at 28 weeks' gestation and within 72 hours of delivery to prevent sensitisation.

Worked Example: Blood Transfusion Compatibility

A patient with blood type B- (B negative, meaning Rh D negative) requires a blood transfusion. The blood bank has the following units available: A+, A-, B+, B-, AB+, AB-, O+, O-. (a) Which units can be safely transfused? (b) Explain the basis for compatibility. (c) Why is Rh compatibility particularly important for women of childbearing age?

Solution

(a) Compatible units for a B- recipient:

ABO: the recipient has anti-A antibodies. Only B or O red blood cells are compatible (no A antigen).
Rh: the recipient is Rh-negative and may have anti-D antibodies if previously sensitised. Only Rh-negative units are safe.
Compatible: B- and O-.

(b) ABO compatibility: the recipient's anti-A antibodies would attack any donor red blood cells with the A antigen (types A and AB), causing a transfusion reaction (agglutination, complement activation, haemolysis). Type B has the B antigen (no reaction with anti-A). Type O has neither A nor B antigens (universal donor for ABO).

Rh compatibility: if the recipient has been previously sensitised to the D antigen (via pregnancy, transfusion, or trauma), their anti-D antibodies would attack Rh+ donor cells. Even if not sensitised, transfusion with Rh+ blood could sensitise the recipient for future pregnancies.

If an Rh-negative woman receives Rh+ blood, she will be sensitised to the D antigen.
In a subsequent pregnancy with an Rh-positive fetus, her anti-D IgG antibodies can cross the placenta and destroy fetal red blood cells (haemolytic disease of the newborn).
This can be prevented by using only Rh-negative blood products for Rh-negative women of childbearing age.

1. Overview of the Immune System​

2. Innate Immunity​

First-Line Defences (Physical and Chemical Barriers)​

Second-Line Defences (Internal Non-Specific Responses)​

Phagocytosis​

Inflammation​

The Complement System​

Natural Killer (NK) Cells​

Interferons​

3. Adaptive Immunity: Overview​

4. Antigen Presentation​

MHC Class I (Endogenous Pathway)​

MHC Class II (Exogenous Pathway)​

5. T-Cell Activation and Response​

Helper T Cells (CD4+\mathrm{CD4}^+CD4+)​

Cytotoxic T Cells (CD8+\mathrm{CD8}^+CD8+)​

Memory T Cells​

6. B-Cell Activation and Humoral Immunity​

B-Cell Activation​

B-Cell Differentiation​

Affinity Maturation and Class Switching​

7. Antibody Structure and Classes​

Antibody (Immunoglobulin) Structure​

Antibody Classes​

Antibody Functions (Effector Mechanisms)​

8. Immunological Memory​

Primary and Secondary Responses​

Types of Immunity​

9. Vaccination​

Principles​

Types of Vaccines​

Vaccine Considerations​

10. ELISA (Enzyme-Linked Immunosorbent Assay)​

Direct ELISA (detecting antigen)​

Indirect ELISA (detecting antibody --- e.g., HIV testing)​

Sandwich ELISA (detecting antigen with higher sensitivity)​

11. Monoclonal Antibodies​

Production (Kohler and Milstein, 1975)​

Applications​

12. Organ Transplantation​

Types of Transplants​

Transplant Rejection​

Immunosuppression​

Tissue Typing and Cross-Matching​

13. HIV/AIDS​

HIV Structure and Life Cycle​

Immunological Effects​

Transmission and Prevention​

Antiretroviral Therapy (ART)​

14. Autoimmune Diseases​

Mechanisms of Loss of Self-Tolerance​

Examples of Autoimmune Diseases​

15. Allergies (Type I Hypersensitivity)​

Mechanism (Sensitisation and Challenge)​

Symptoms​

Anaphylaxis​

Desensitisation Therapy (Allergen Immunotherapy)​

16. Other Immune Disorders​

Immunodeficiency​

Type II Hypersensitivity (Antibody-Mediated)​

Type III Hypersensitivity (Immune Complex-Mediated)​

Type IV Hypersensitivity (Delayed-Type, T-Cell Mediated)​

Common Pitfalls​

Practice Problems​

Worked Examples​

Common Pitfalls (Expanded)​

Exam-Style Problems​

If You Get These Wrong, Revise:​

Additional Worked Examples​

Additional Common Pitfalls​

Additional Exam-Style Problems with Full Solutions​

Cross-References to Related Topics​

Supplementary: Antibody Diversity -- V(D)J Recombination (HL Extension)​

The Challenge of Antibody Diversity​

Antibody Structure Recap​

V(D)J Recombination​

Sources of Antibody Diversity​

Worked Example: Calculating Antibody Diversity​

The Major Histocompatibility Complex (MHC)​

T Cell Development and Selection​

1. Overview of the Immune System

2. Innate Immunity

First-Line Defences (Physical and Chemical Barriers)

Second-Line Defences (Internal Non-Specific Responses)

Phagocytosis

Inflammation

The Complement System

Natural Killer (NK) Cells

Interferons

3. Adaptive Immunity: Overview

4. Antigen Presentation

MHC Class I (Endogenous Pathway)

MHC Class II (Exogenous Pathway)

5. T-Cell Activation and Response

Helper T Cells ( $\mathrm{CD4}^+$ )

Cytotoxic T Cells ( $\mathrm{CD8}^+$ )

Memory T Cells

6. B-Cell Activation and Humoral Immunity

B-Cell Activation

B-Cell Differentiation

Affinity Maturation and Class Switching

7. Antibody Structure and Classes

Antibody (Immunoglobulin) Structure

Antibody Classes

Antibody Functions (Effector Mechanisms)

8. Immunological Memory

Primary and Secondary Responses

Types of Immunity

9. Vaccination

Principles

Types of Vaccines

Vaccine Considerations

10. ELISA (Enzyme-Linked Immunosorbent Assay)

Direct ELISA (detecting antigen)

Indirect ELISA (detecting antibody --- e.g., HIV testing)

Sandwich ELISA (detecting antigen with higher sensitivity)

11. Monoclonal Antibodies

Production (Kohler and Milstein, 1975)

Applications

12. Organ Transplantation

Types of Transplants

Transplant Rejection

Immunosuppression

Tissue Typing and Cross-Matching

13. HIV/AIDS

HIV Structure and Life Cycle

Immunological Effects

Transmission and Prevention

Antiretroviral Therapy (ART)

14. Autoimmune Diseases

Mechanisms of Loss of Self-Tolerance

Examples of Autoimmune Diseases

15. Allergies (Type I Hypersensitivity)

Mechanism (Sensitisation and Challenge)

Symptoms

Anaphylaxis

Desensitisation Therapy (Allergen Immunotherapy)

16. Other Immune Disorders

Immunodeficiency

Type II Hypersensitivity (Antibody-Mediated)

Type III Hypersensitivity (Immune Complex-Mediated)

Type IV Hypersensitivity (Delayed-Type, T-Cell Mediated)

Common Pitfalls

Practice Problems

Worked Examples

Common Pitfalls (Expanded)

Exam-Style Problems

If You Get These Wrong, Revise:

Additional Worked Examples

Additional Common Pitfalls

Additional Exam-Style Problems with Full Solutions

Cross-References to Related Topics

Supplementary: Antibody Diversity -- V(D)J Recombination (HL Extension)

The Challenge of Antibody Diversity

Antibody Structure Recap

V(D)J Recombination

Sources of Antibody Diversity

Worked Example: Calculating Antibody Diversity

The Major Histocompatibility Complex (MHC)

T Cell Development and Selection