17.10.02 · mol-cell-bio / immunology

Adaptive immunity overview: B cells, T cells, clonal selection, and the antibody response

stub3 tiersLean: nonepending prereqs

Anchor (Master): Janeway's Immunobiology, 10th ed. (2022)

Intuition Beginner

The innate immune system (Unit 17.10.01) attacks anything that looks generally foreign. But some pathogens evade those broad defenses. The body needs a second system that can learn to recognise specific invaders and remember them for years. This is the adaptive immune system — so named because it adapts to each new threat it encounters.

Two types of white blood cells carry out adaptive immunity. B cells produce antibodies — Y-shaped proteins that stick to specific molecules on the pathogen's surface, flagging it for destruction. Each B cell makes antibodies with one particular shape, capable of binding one particular target (called an antigen). T cells take a different approach: some travel to sites of infection and kill cells that have been hijacked by viruses from the inside, while others act as coordinators that help B cells and other immune cells do their jobs more effectively.

The first time you encounter a new pathogen, only a few of your B and T cells can recognise it. The response starts slowly and takes days to build. But those cells multiply rapidly, and after the infection is cleared, a population of long-lived memory cells remains. If the same pathogen returns months or years later, these memory cells launch a faster, stronger attack — often clearing it before you feel sick. This is why vaccines work: they expose you to a harmless version of the pathogen so that memory cells form without you having to endure the real disease.

Visual Beginner

The diagram shows the adaptive immune response in sequence. A pathogen enters the body and is picked up by a dendritic cell, which carries it to a lymph node. There, the dendritic cell presents fragments of the pathogen to T cells. Helper T cells that match the antigen become activated and proliferate. Activated helper T cells then stimulate B cells that recognise the same antigen. The B cells multiply and differentiate into plasma cells that secrete large amounts of antibody, and into memory B cells that persist for long-term protection. Meanwhile, cytotoxic T cells that recognise infected host cells proliferate and kill their targets directly.

Worked example Beginner

Consider what happens when you get infected with influenza virus for the first time.

Day 0. You inhale influenza virus particles. They infect epithelial cells lining your airway. The innate immune system responds within hours: macrophages and neutrophils arrive, complement is activated, and interferons are released. But the virus replicates faster than innate immunity can contain it.

Day 1-2. Dendritic cells in the lung tissue swallow virus particles, process them into protein fragments, and travel to nearby lymph nodes. In the lymph node, each dendritic cell displays these fragments on its surface for T cells to inspect. Out of millions of T cells in the lymph node, a small number happen to carry receptors that fit the influenza fragments. These matching T cells bind tightly, become activated, and begin dividing rapidly — this is clonal selection.

Day 3-5. The activated helper T cells stimulate B cells that also recognise influenza antigens. The B cells proliferate and differentiate into plasma cells that secrete anti-influenza antibodies. These antibodies travel through the bloodstream to the lungs, bind to virus particles, and mark them for destruction. Cytotoxic T cells travel to the infected lung tissue and kill epithelial cells that are producing new virus.

Day 7-10. The combined antibody and T cell response clears the infection. Most of the expanded B and T cells die off, but a fraction survive as memory cells — long-lived cells that remain in your body for years.

Next exposure. If you encounter influenza again, your memory B and T cells respond within 1-2 days instead of 5-7. The antibody response is faster, stronger, and more precise. You may fight off the virus before symptoms develop.

Check your understanding Beginner

Exercise (easy, multiple choice).

What is the primary advantage of memory B and T cells compared to naive (never-activated) lymphocytes?

A. Memory cells can produce antibodies without T cell help. B. Memory cells respond faster and in greater numbers upon re-exposure to the same antigen. C. Memory cells can recognise a wider range of pathogens. D. Memory cells live forever without any metabolic activity.

Hint

Think about why you do not get sick from the same strain of chickenpox twice, even decades after the first infection.

Answer

B. Memory cells respond faster and in greater numbers upon re-exposure. During the primary response, only a handful of naive lymphocytes match the antigen, and they need days to proliferate. Memory cells are already expanded in number and require less stimulation to reactivate. They do not recognise a wider range of pathogens (each memory cell is specific for one antigen), and they do not live forever (though some persist for decades).

Formal definition Intermediate+

Adaptive immunity is the antigen-specific immune response mediated by B lymphocytes (which secrete antibodies) and T lymphocytes (which kill infected cells and regulate immune responses). Unlike innate immunity, adaptive immunity exhibits specificity (each lymphocyte recognises one particular antigen), diversity (the repertoire can recognise an estimated $1 0^{11}$ or more distinct antigens), memory (re-exposure produces a faster, stronger response), and self/non-self discrimination (lymphocytes reactive against the body's own molecules are normally eliminated during development).

B cell development

B cells develop in the bone marrow from hematopoietic stem cells. Development proceeds through defined stages marked by sequential rearrangement of immunoglobulin gene segments:

Pro-B cell. The heavy-chain locus undergoes D-to-J recombination, followed by V-to-DJ recombination, catalysed by the RAG1/RAG2 (recombination-activating gene) enzymes and TdT (terminal deoxynucleotidyl transferase, which adds N-nucleotides at junctions for additional diversity). If a functional heavy chain is produced, it pairs with surrogate light chain to form the pre-B cell receptor, signaling successful rearrangement and triggering proliferation.

Pre-B cell. Light-chain loci (kappa, then lambda if kappa rearrangement fails) undergo V-to-J recombination. A successfully rearranged light chain pairs with the heavy chain to form a complete IgM surface immunoglobulin. Allelic exclusion ensures that each B cell expresses only one heavy chain and one light chain — a single antigen specificity.

Negative selection. Immature B cells that bind strongly to self-antigens present in the bone marrow are eliminated by one of three mechanisms: clonal deletion (apoptosis), receptor editing (secondary light-chain rearrangement to change specificity), or anergy (functional inactivation). This is the first checkpoint for self-tolerance.

Mature naive B cells express both IgM and IgD on their surface (produced by alternative splicing of the same heavy-chain transcript) and circulate through blood and lymphoid organs searching for their cognate antigen.

T cell development

T cell precursors migrate from the bone marrow to the thymus, where they undergo a stringent selection process:

Double-negative stage. Early thymocytes lack both CD4 and CD8 co-receptors. The TCR beta chain undergoes V-D-J rearrangement (analogous to immunoglobulin heavy-chain rearrangement). Successful beta-chain production leads to beta-selection: the beta chain pairs with pre-T alpha to form the pre-TCR, triggering proliferation and progression to the double-positive stage.

Double-positive stage. Thymocytes express both CD4 and CD8 and begin TCR alpha-chain rearrangement (V-to-J). The resulting alpha-beta TCR is tested against self-peptide complexes presented by thymic epithelial cells.

Positive selection. Thymocytes whose TCR binds self-MHC (with moderate affinity) receive a survival signal. Thymocytes that cannot bind any self-MHC die by neglect (no survival signal). This ensures that mature T cells are restricted to recognising antigen presented by the host's own MHC molecules. CD4 or CD8 lineage commitment occurs at this stage: TCRs that bind MHC class II become CD4+ helper T cells; TCRs that bind MHC class I become CD8+ cytotoxic T cells.

Negative selection. Thymocytes whose TCR binds self-peptide with high affinity are eliminated by apoptosis. This removes strongly self-reactive T cells and is the central mechanism of central tolerance. The AIRE (autoimmune regulator) transcription factor in medullary thymic epithelial cells drives expression of tissue-specific antigens in the thymus, ensuring that T cells are tested against proteins from throughout the body (not just thymic proteins).

Approximately 98% of thymocytes die during selection — only 2% survive both positive and negative selection to emigrate as mature naive T cells.

Clonal selection theory

The clonal selection theory, proposed by Macfarlane Burnet (1957), is the foundational principle of adaptive immunity. It states that:

Each lymphocyte expresses receptors with a single antigen specificity, determined randomly during lymphocyte development (before any encounter with antigen).
The mature immune system contains a vast repertoire of lymphocytes, each with a unique receptor.
When an antigen enters the body, it binds to and activates only those lymphocytes whose receptors match it.
The activated lymphocytes proliferate extensively (clonal expansion), producing a large population of effector cells and memory cells, all sharing the same antigen specificity.
After the antigen is cleared, most effector cells die, but memory cells persist.

This theory explains specificity (only matching clones respond), diversity (the pre-existing repertoire covers vast antigenic space), and memory (the expanded clone provides faster future responses). The random generation of receptor diversity also explains how the adaptive immune system can respond to antigens it has never encountered in the evolutionary history of the species — including synthetic molecules that do not exist in nature.

Antibody structure and isotypes

Antibodies (immunoglobulins, Ig) are Y-shaped proteins composed of two identical heavy chains and two identical light chains, linked by disulfide bonds. Each chain has a variable (V) region at its tip that forms the antigen-binding site and a constant (C) region that determines the effector function.

Five heavy-chain isotypes define the antibody classes:

Isotype	Structure	Primary function
IgM	Pentamer (5 Y units + J chain)	First antibody produced in primary response; efficient complement activator
IgG	Monomer	Most abundant in serum; neutralises toxins, opsonises pathogens, crosses placenta
IgA	Dimer (2 Y units + J chain) at mucosal surfaces	Mucosal immunity; secreted in saliva, tears, breast milk
IgE	Monomer	Binds mast cells and basophils; mediates allergic and anti-parasite responses
IgD	Monomer	Co-expressed with IgM on naive B cell surface; function not fully understood

Class (isotype) switching. Activated B cells can change the constant region of their antibody from IgM to IgG, IgA, or IgE through a DNA recombination event called class switch recombination (CSR), while retaining the same variable region (and therefore the same antigen specificity). CSR is directed by cytokines from helper T cells: IL-4 promotes switching to IgG1 and IgE; IFN-gamma promotes IgG2a; TGF-beta promotes IgA. This allows the same antigen specificity to be deployed with different effector functions appropriate to the type of infection.

Primary versus secondary antibody response

Primary response. Upon first exposure to an antigen, naive B cells require T cell help to become activated. The lag phase is 5-10 days. IgM appears first, followed by a small IgG response. Total antibody levels are moderate.

Secondary response. Upon re-exposure, memory B cells respond within 1-3 days. The response is larger (10-100 fold higher antibody titre), predominantly IgG (due to prior class switching), and the antibodies have higher affinity for the antigen (due to affinity maturation in the germinal center during the primary response). This difference is the basis of booster vaccinations.

T cell activation: the two-signal model

Naive T cells require two simultaneous signals for activation:

Signal 1. The T cell receptor (TCR) binds its cognate peptide presented on MHC (MHC class I for CD8+ T cells, MHC class II for CD4+ T cells) on the surface of an antigen-presenting cell (APC).

Signal 2 (co-stimulation). CD28 on the T cell binds B7 (CD80/CD86) on the APC. Without this second signal, TCR engagement alone leads to anergy (functional inactivation) rather than activation.

This two-signal requirement is a safety mechanism: it ensures that T cells are only activated when both antigen (signal 1) and an inflammatory context indicating genuine danger (signal 2, upregulated on APCs by innate immune signals) are present.

Counterexamples to common slips

Antibodies are always beneficial. In autoimmune diseases (lupus, rheumatoid arthritis), antibodies attack self-tissues. In allergies, IgE antibodies recognise harmless environmental antigens (pollen, peanut proteins) and trigger mast-cell degranulation, causing symptoms ranging from mild itching to fatal anaphylaxis.
T cells only kill infected cells. CD4+ helper T cells do not kill targets directly. They secrete cytokines that coordinate the immune response: Th1 cells activate macrophages; Th2 cells help B cells produce antibodies; Th17 cells recruit neutrophils; Treg cells suppress immune responses to prevent autoimmunity.
Clonal selection only applies to B cells. Clonal selection operates for both B and T lymphocytes. In both lineages, a receptor is generated randomly before antigen encounter, and antigen-driven selection expands the matching clone.

Key mechanism Intermediate+

Mechanism: Clonal selection and the germinal center reaction.

The germinal center is the anatomical site where B cell clonal selection, affinity maturation, and class switch recombination occur. It forms in lymphoid follicles 4-7 days after antigen exposure and operates as a Darwinian selection machine for antibody-producing cells.

Step 1: Formation. Activated B cells migrate into B cell follicles in lymph nodes and form the germinal center. The germinal center has two zones: the dark zone (where B cells proliferate rapidly) and the light zone (where selection occurs).

Step 2: Proliferation and somatic hypermutation. In the dark zone, B cells undergo rapid cell division (every 6-12 hours). During each division, the enzyme activation-induced cytidine deaminase (AID) introduces point mutations into the variable region of the immunoglobulin genes at a rate approximately one million times higher than the normal spontaneous mutation rate. This process, called somatic hypermutation, generates variant B cells with slightly altered antigen-binding sites. Most mutations reduce or abolish binding; a small fraction increase the affinity for the antigen.

Step 3: Selection. B cells migrate from the dark zone to the light zone, where they encounter follicular dendritic cells (FDCs) that display antigen on their surface as immune complexes. B cells whose B cell receptors bind antigen with sufficient affinity capture the antigen, process it, and present peptide fragments on MHC class II to follicular helper T cells (Tfh). Tfh cells that recognise the same antigen provide survival signals (CD40 ligand binding CD40 on the B cell, plus cytokines IL-21 and IL-4). B cells that fail to capture antigen or receive T cell help die by apoptosis within hours.

Step 4: Cyclic re-entry. Selected B cells can re-enter the dark zone for further rounds of proliferation and somatic hypermutation, followed by another round of selection in the light zone. Each cycle increases the average affinity of the B cell population for the antigen — this is affinity maturation.

Step 5: Differentiation. After several cycles, high-affinity B cells differentiate into either plasma cells (long-lived antibody-secreting cells that migrate to the bone marrow) or memory B cells (recirculating cells that provide rapid secondary responses). The decision between plasma cell and memory cell fate is influenced by the strength of T cell help and the affinity of the B cell receptor.

The germinal center reaction is a nontrivial example of Darwinian evolution operating within a single organism over days rather than millennia. Mutation (AID-mediated somatic hypermutation), selection (competition for limited antigen and T cell help), and heritable variation (the immunoglobulin gene mutation is passed to daughter cells) are all present. The output is a population of B cells whose antibodies bind the antigen with affinities that can be 10- to 1000-fold higher than those of the original naive B cell.

Exercises Intermediate+

Exercise 3 (medium, symbolic).

Explain the two-signal model of T cell activation. Why is signal 2 (co-stimulation) required in addition to signal 1 (TCR engagement)? What happens to a T cell that receives signal 1 without signal 2?

Hint

Consider what would happen if T cells could be activated by antigen alone, without any check on whether the antigen was presented in an inflammatory context.

Answer

Signal 1 is TCR binding to peptide on an antigen-presenting cell. Signal 2 is CD28 on the T cell binding B7 (CD80/CD86) on the APC. Both signals are required for full T cell activation, proliferation, and cytokine production.

The requirement for co-stimulation is a safety mechanism. B7 molecules are upregulated on APCs only when the innate immune system has detected danger (PAMPs, DAMPs, inflammatory cytokines). If a T cell encounters its antigen on a healthy cell lacking B7 (which is the normal state of most non-immune cells), signal 1 without signal 2 induces anergy — a state of long-lasting functional unresponsiveness in which the T cell cannot be activated even by subsequent full stimulation. This prevents T cells from responding to self-antigens presented by healthy tissues.

The clinical relevance: CTLA-4 (cytotoxic T lymphocyte antigen 4) is a competitive inhibitor of CD28 that binds B7 with higher affinity, terminating co-stimulation. Ipilimumab, an anti-CTLA-4 antibody used in cancer immunotherapy, blocks this checkpoint, sustaining T cell activation against tumor antigens.

Exercise 5 (medium, numeric).

The human TCR beta locus contains approximately 52 V gene segments, 2 D gene segments, and 13 J gene segments. If combinatorial diversity (V-D-J rearrangement) were the only source of diversity, and assuming random pairing with approximately 70 alpha-chain V segments and 61 alpha-chain J segments, calculate the approximate combinatorial diversity of the alpha-beta TCR repertoire from rearrangement alone (ignoring junctional diversity from N-additions).

Hint

Multiply the number of possible beta chains (V times D times J combinations) by the number of possible alpha chains (V times J combinations). This gives the number of distinct alpha-beta pairs.

Answer

Beta-chain combinations: $52 \times 2 \times 13 = 1352$ . Alpha-chain combinations: $70 \times 61 = 4270$ . Total combinatorial diversity: $1352 \times 4270 \approx 5.8 \times 1 0^{6}$ distinct alpha-beta TCR pairs.

This is a vast underestimate of the true TCR repertoire diversity (estimated at $1 0^{11}$ or more) because it ignores junctional diversity: TdT adds random N-nucleotides at the V-D and D-J junctions of the beta chain and at the V-J junction of the alpha chain, and exonucleases trim gene segment ends before joining. Junctional diversity contributes at least $1 0^{11}$ additional combinations, dwarfing the combinatorial contribution.

Exercise 6 (hard, symbolic).

Explain how AIRE (autoimmune regulator) in medullary thymic epithelial cells (mTECs) contributes to central tolerance. What is the consequence of AIRE deficiency in humans?

Hint

Most proteins expressed in the thymus are thymus-specific. But T cells need to be tested against proteins from the pancreas, thyroid, brain, and other organs. How does the thymus gain access to these tissue-specific proteins?

Answer

AIRE is a transcription factor expressed in mTECs that drives ectopic expression of tissue-specific antigens — proteins that are normally restricted to specific organs (insulin from pancreatic beta cells, thyroglobulin from the thyroid, myelin basic protein from the nervous system, and so on). By expressing these proteins in the thymus, mTECs present them to developing T cells during negative selection. T cells whose TCRs bind strongly to these tissue-specific self-antigens are deleted before leaving the thymus.

AIRE deficiency causes autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also called autoimmune polyglandular syndrome type 1. Without AIRE, tissue-specific antigens are not presented in the thymus, self-reactive T cells escape negative selection, and patients develop multiple autoimmune diseases: chronic mucocutaneous candidiasis, hypoparathyroidism, Addison disease, type 1 diabetes, hepatitis, and others. The disease demonstrates that central tolerance requires active presentation of tissue-specific self-antigens in the thymus, not merely the absence of foreign antigen.

Exercise 7 (hard, symbolic).

Describe the germinal center reaction in detail, including the roles of the dark zone, light zone, AID, follicular dendritic cells, and Tfh cells. Explain how this process produces antibodies with higher affinity than those generated by the initial naive B cell.

Hint

The germinal center is a selection machine. B cells mutate their receptors, then compete for survival signals. Only the best binders survive each round.

Answer

The germinal center reaction operates as iterative rounds of mutation and selection:

Dark zone: Activated B cells (called centroblasts) proliferate rapidly. During each cell division, AID deaminates cytidine to uridine in the variable regions of the immunoglobulin heavy-chain and light-chain genes. This triggers error-prone repair pathways (base excision repair and mismatch repair) that introduce point mutations at a rate of approximately $1 0^{- 3}$ per base pair per division — roughly $1 0^{6}$ times the background mutation rate. The mutations are focused on the complementarity-determining regions (CDRs) that form the antigen-binding site.

Light zone: After several divisions, B cells (now called centrocytes) migrate to the light zone and encounter antigen displayed as immune complexes on follicular dendritic cells (FDCs). FDCs retain antigen in its native conformation on their surface for weeks to months. B cells compete for limited antigen: those with higher-affinity B cell receptors capture more antigen, process it, and present more peptide on MHC class II.

Tfh selection: Follicular helper T cells (Tfh) in the light zone examine the peptide complexes on centrocytes. Only B cells that present sufficient antigen-derived peptide receive Tfh help: CD40 ligand (binding CD40 on the B cell) and cytokines (IL-21, IL-4). This help delivers anti-apoptotic signals and promotes further differentiation. B cells that fail to capture antigen or receive Tfh help undergo apoptosis within 6-12 hours.

Cyclic re-entry: Selected centrocytes can re-enter the dark zone for additional rounds of proliferation and somatic hypermutation. Each cycle increases the average affinity: beneficial mutations (improved binding) are positively selected, while deleterious mutations (reduced binding) lead to cell death. After 5-20 cycles over 2-3 weeks, antibodies can reach affinities of $1 0^{- 10}$ to $1 0^{- 12}$ M (dissociation constants), compared to $1 0^{- 6}$ to $1 0^{- 7}$ M for the original naive B cell receptor.

Output: High-affinity B cells exit the germinal center as either long-lived plasma cells (migrating to the bone marrow to secrete antibody for years) or memory B cells (recirculating and providing rapid secondary responses).

Somatic hypermutation, TCR signaling, and immune checkpoints Master

AID and the mechanism of somatic hypermutation

Activation-induced cytidine deaminase (AID, encoded by AICDA) is the enzyme that initiates both somatic hypermutation and class switch recombination. AID deaminates cytidine residues in single-stranded DNA to uridine, creating U mismatches. These lesions are processed by three competing repair pathways, each producing a different mutagenic outcome:

Replication pathway. If the uridine is replicated before repair, DNA polymerase reads it as thymidine, producing a C-to-T transition (or G-to-A on the complementary strand). This accounts for a large fraction of observed somatic hypermutations.

Base excision repair (BER). Uracil-DNA glycosylase (UNG) removes the uracil, creating an abasic site. Error-prone translesion polymerases (Pol eta, Pol iota, Pol zeta) fill in across the abasic site, introducing mutations at both the deaminated position and neighbouring bases. Rev1 (a cytidyl transferase) is particularly important for generating C-to-G transversions.

Mismatch repair (MSH). The MSH2/MSH6 heterodimer recognises the U mismatch and recruits error-prone polymerase Pol eta to the site. Pol eta preferentially introduces A-to-T and T-to-A transversions at A base pairs surrounding the initial lesion, explaining the characteristic mutation pattern of somatic hypermutation (hotspots at WRC motifs, where W = A/T, R = A/G, C = the deaminated base).

Targeting. AID activity is focused on the variable regions of immunoglobulin genes. Targeting is mediated by transcription through the immunoglobulin locus (which generates the single-stranded DNA substrate), enhancer elements (the intronic enhancer Ei and the 3-prime regulatory region), and histone modifications (H3K4me3 marks recruit AID through its interaction with the Spt6 elongation factor). Off-target AID activity at non-immunoglobulin loci (particularly BCL6, c-MYC, and PAX5) is a source of chromosomal translocations in B cell lymphomas — the t(14;18) translocation in follicular lymphoma and the t(8;14) translocation in Burkitt lymphoma both arise from aberrant AID activity during the germinal center reaction.

Class switch recombination

Class switch recombination replaces the constant region of the immunoglobulin heavy chain (from $C_{μ}$ to $C_{γ}$ , $C_{α}$ , or $C_{ϵ}$ ) while preserving the variable region. The mechanism involves AID-mediated deamination in switch (S) regions — repetitive, GC-rich DNA sequences upstream of each constant region gene. The processing of AID-generated lesions generates double-strand breaks in two switch regions (e.g., $S_{μ}$ and $S_{γ} 1$ for switching to IgG1). The DNA between the two breaks is excised as a circular DNA fragment (a "switch circle"), and the free ends are joined by the non-homologous end joining (NHEJ) DNA repair pathway.

CSR requires activation-induced cytidine deaminase, UNG, MSH2/MSH6, and the NHEJ machinery (Ku70/Ku80, DNA-PKcs, XRCC4, DNA ligase IV). Germline transcription through the target switch region (directed by cytokine signals) opens the chromatin and provides the single-stranded DNA substrate for AID. Different cytokines activate germline transcription of different switch regions: IL-4 activates $S_{ϵ}$ (switching to IgE) and $S_{γ} 1$ (switching to IgG1); IFN-gamma activates $S_{γ} 2 a$ (switching to IgG2a); TGF-beta activates $S_{α}$ (switching to IgA).

TCR signaling cascade

T cell receptor signaling begins when the TCR/CD3 complex binds peptide on an antigen-presenting cell. The signaling cascade converts this extracellular recognition event into intracellular gene expression changes through a well-characterised kinase cascade.

Lck and ZAP-70. The Src-family kinase Lck (p56) is constitutively associated with CD4 and CD8 co-receptors. When the co-receptor binds MHC, Lck is brought into proximity with the CD3 ITAMs (immunoreceptor tyrosine-based activation motifs). Lck phosphorylates the ITAMs on CD3 zeta and epsilon chains. Phosphorylated ITAMs recruit the Syk-family kinase ZAP-70 (zeta-chain-associated protein kinase of 70 kDa) via its SH2 domains. Lck then phosphorylates and activates ZAP-70.

LAT and SLP-76. Active ZAP-70 phosphorylates the transmembrane adaptor LAT (linker for activation of T cells) on multiple tyrosine residues. Phosphorylated LAT serves as a scaffold that recruits the cytosolic adaptor SLP-76 (via the Gads adaptor protein), PLC-gamma1 (via Grb2), and other signaling molecules. The LAT signalosome integrates multiple downstream pathways:

PLC-gamma1 pathway: PLC-gamma1 hydrolyses PIP2 into IP3 (which releases calcium from the ER, activating the phosphatase calcineurin, which dephosphorylates NFAT, allowing it to enter the nucleus) and DAG (which activates PKC-theta and the Ras-GRP/Ras/MEK/ERK pathway, leading to AP-1 transcription factor activation). NFAT and AP-1 cooperate to activate IL-2 transcription — the key growth factor for T cell clonal expansion.
Vav/Rac pathway: Phosphorylated SLP-76 recruits Vav (a guanine nucleotide exchange factor), which activates Rac and Cdc42, leading to actin cytoskeletal reorganisation and immunological synapse formation.
PI3K/Akt pathway: CD28 co-stimulation activates PI3K, generating PIP3, which recruits and activates Akt (PKB). Akt promotes cell survival (via mTOR and Bcl-2 family regulation) and metabolic reprogramming (glycolysis upregulation to support rapid proliferation).

Immunological synapse. T cell activation occurs at a specialised cell-cell contact called the immunological synapse (also called the supramolecular activation cluster, SMAC). The synapse has a centripetal organisation: TCRs and peptide complexes accumulate in the central SMAC (cSMAC), surrounded by a ring of adhesion molecules (LFA-1/ICAM-1) forming the peripheral SMAC (pSMAC). The synapse serves as a stable signaling platform that sustains TCR signaling for hours, which is required for full T cell activation. Sustained signaling (rather than transient contact) is the critical variable determining whether a T cell becomes an effector cell, an anergic cell, or a memory cell.

Co-stimulation and immune checkpoint pathways

The balance between activating and inhibitory signals at the T cell surface determines the magnitude and duration of the adaptive immune response.

CD28-B7 activating pathway. CD28 on naive T cells binds B7-1 (CD80) and B7-2 (CD86) on activated APCs. B7 expression is upregulated on APCs by innate immune signals (TLR engagement, inflammatory cytokines). CD28 signaling enhances IL-2 production, promotes cell survival (via Bcl-xL upregulation), and increases glucose metabolism to support clonal expansion. CD28 is the primary co-stimulatory receptor for naive T cell activation.

CTLA-4 checkpoint. CTLA-4 (CD152) is upregulated on T cells 2-3 days after activation. CTLA-4 binds B7 with approximately 20-fold higher affinity than CD28 and delivers an inhibitory signal that terminates T cell activation. CTLA-4 also removes B7 from the APC surface by trans-endocytosis — CTLA-4 physically extracts B7 molecules from the APC membrane and internalises them, reducing the availability of co-stimulation for other T cells. CTLA-4 knockout mice develop fatal lymphoproliferative disease, demonstrating that this checkpoint is essential for immune homeostasis.

PD-1/PD-L1 checkpoint. Programmed death-1 (PD-1) is expressed on activated T cells and binds its ligands PD-L1 and PD-L2 (expressed on many tissues and some immune cells). PD-1 engagement recruits the phosphatases SHP-1 and SHP-2 to its cytoplasmic tail, which dephosphorylate key signaling molecules in the TCR and CD28 pathways, dampening T cell activation. Unlike CTLA-4 (which primarily regulates the initial activation in lymph nodes), PD-1 primarily regulates T cell activity in peripheral tissues during the effector phase. Chronic antigen exposure (as in persistent viral infections and cancer) leads to sustained PD-1 expression and a state called T cell exhaustion, in which T cells lose effector function progressively.

Checkpoint immunotherapy. Antibodies blocking CTLA-4 (ipilimumab) and PD-1/PD-L1 (nivolumab, pembrolizumab, atezolizumab) have transformed cancer treatment. By releasing inhibitory brakes on T cells, these agents enable the adaptive immune system to recognise and destroy tumor cells. The 2018 Nobel Prize in Physiology or Medicine was awarded to James Allison (CTLA-4) and Tasuku Honjo (PD-1) for this work. Response rates vary by tumor type (highest in melanoma, lung cancer, and Hodgkin lymphoma), and immune-related adverse events (colitis, pneumonitis, thyroiditis) reflect the breakdown of self-tolerance that occurs when checkpoints are blocked.

Helper T cell subsets

Naive CD4+ T cells differentiate into distinct effector subsets, each producing a characteristic cytokine profile and directing a specific type of immune response. The subset decision is determined by the cytokine environment during initial activation:

Th1 cells. Differentiated by IL-12 and IFN-gamma. Produce IFN-gamma, which activates macrophages to kill intracellular bacteria and parasites. Th1 cells are critical for defence against Mycobacterium tuberculosis and other intracellular pathogens. The transcription factor T-bet (encoded by TBX21) is the master regulator of Th1 differentiation.

Th2 cells. Differentiated by IL-4. Produce IL-4, IL-5, and IL-13, which promote eosinophil recruitment, IgE class switching, mucus production, and goblet cell hyperplasia. Th2 cells coordinate defence against extracellular parasites (helminths) and are also responsible for allergic responses. The transcription factor GATA3 is the master regulator.

Th17 cells. Differentiated by TGF-beta plus IL-6 (in mice) or IL-1beta plus IL-23 (in humans). Produce IL-17A, IL-17F, and IL-22, which recruit neutrophils and stimulate epithelial production of antimicrobial peptides. Th17 cells are critical for defence against extracellular bacteria and fungi at mucosal surfaces. The transcription factor RORgammat is the master regulator. Th17 dysregulation is implicated in autoimmune diseases: psoriasis, ankylosing spondylitis, and inflammatory bowel disease.

Tfh cells. Follicular helper T cells are distinguished by expression of CXCR5 (which directs migration into B cell follicles) and ICOS (inducible co-stimulator). Tfh cells produce IL-21 and provide help to B cells in the germinal center, driving affinity maturation, class switching, and plasma cell differentiation. The transcription factor Bcl-6 is the master regulator. Loss-of-function mutations in ICOS or SAP (SLAM-associated protein, required for Tfh-B cell cognate interaction) cause antibody deficiency syndromes in humans.

Treg cells. Regulatory T cells suppress immune responses and maintain self-tolerance. Natural Tregs (nTreg) develop in the thymus; induced Tregs (iTreg) differentiate from naive CD4+ T cells in the periphery under the influence of TGF-beta alone (without the inflammatory signals that drive Th17 differentiation). Both populations require the transcription factor Foxp3 (forkhead box P3). Tregs suppress other T cells through multiple mechanisms: secretion of inhibitory cytokines (IL-10, TGF-beta), consumption of IL-2 (starving effector T cells), CTLA-4-mediated removal of B7 from APCs, and direct cytolysis. Loss-of-function mutations in FOXP3 cause IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) — a severe, often fatal autoimmune disease affecting multiple organs.

The plasticity of helper T cell subsets is an active area of research. Under certain conditions, differentiated Th cells can convert to other subsets (e.g., Th1 to Th17, or Treg to Th17), particularly in inflammatory environments. This plasticity complicates the simple subset model but reflects the adaptability of CD4+ T cells in responding to changing pathogen challenges.

Immunological memory

Immunological memory is the defining feature of adaptive immunity. It manifests at both the B cell and T cell levels:

Memory B cells. Generated in the germinal center, memory B cells carry somatically hypermutated, class-switched B cell receptors. They circulate through blood and lymphoid tissues for years to decades. Upon re-exposure to antigen, memory B cells rapidly differentiate into plasma cells (bypassing the need for germinal center re-entry) and produce high-affinity IgG within 1-3 days. Some memory B cells also re-enter germinal centers for additional rounds of affinity maturation upon secondary exposure, further improving antibody quality.

Long-lived plasma cells. A subset of plasma cells generated by the germinal center migrates to the bone marrow, where they occupy survival niches provided by stromal cells and megakaryocytes. These long-lived plasma cells secrete antibody continuously for years without needing further antigen exposure, maintaining a baseline level of protective serum antibody. This is why tetanus vaccination provides protection for 10+ years — bone marrow plasma cells continue to produce anti-tetanus antibodies long after the vaccine antigen has disappeared.

Memory T cells. Memory CD8+ T cells are maintained by homeostatic proliferation driven by IL-7 and IL-15 (cytokines that do not require antigen-specific stimulation). They exist in several subsets: central memory T cells (Tcm, expressing CCR7 and CD62L, residing in lymph nodes, with high proliferative capacity), effector memory T cells (Tem, lacking CCR7, patrolling peripheral tissues, with immediate effector function), and tissue-resident memory T cells (Trm, permanently localised in barrier tissues such as skin, gut, and lung, providing first-response defence at pathogen entry sites). The transcription factors TCF-1 (Tcm) and Blimp-1 (Tem, Trm) regulate memory T cell differentiation.

The maintenance of memory does not require persistent antigen. Early experiments by Zinkernagel and Hengartner (2004) showed that memory T cells survive for the lifespan of the mouse without any detectable antigen, maintained instead by low-level homeostatic cytokine signaling. This has practical implications: memory persists even when the pathogen has been completely eradicated from the body.

Connections Master

Innate immunity 17.10.01. The innate immune system provides the activation signals that initiate adaptive immunity. Dendritic cells activated by PAMPs through TLRs upregulate B7 co-stimulatory molecules and MHC expression, providing both signal 1 and signal 2 for T cell activation. Without innate immune activation, adaptive immunity does not respond. Conversely, adaptive immune effectors (antibodies, activated T cells) direct innate effector mechanisms to specific targets: antibodies activate complement and opsonise pathogens for phagocytosis; Th1-secreted IFN-gamma activates macrophages.
Cell signaling 17.07.01. The TCR signaling cascade (Lck, ZAP-70, LAT, PLC-gamma1, Ras/ERK, NFAT) uses the same molecular logic as RTK and GPCR signaling cascades studied in earlier units: receptor engagement activates a kinase cascade, which produces second messengers (IP3, DAG), which activate transcription factors (NFAT, NF-kB, AP-1). The PI3K/Akt pathway downstream of CD28 co-stimulation was covered in Unit 17.07.03.
DNA repair 17.06.02 pending. V(D)J recombination and class switch recombination both generate double-strand breaks that are repaired by the NHEJ pathway (Ku70/Ku80, DNA-PKcs, XRCC4, DNA ligase IV). Deficiencies in NHEJ components cause severe combined immunodeficiency (SCID) because lymphocytes cannot complete receptor gene rearrangement. Artemis deficiency (a nuclease required for opening hairpin-sealed coding ends during V(D)J recombination) causes RS-SCID (radiation-sensitive SCID).
Apoptosis 17.08.04 pending. Negative selection in the thymus and bone marrow eliminates self-reactive lymphocytes by apoptosis. Activation-induced cell death (AICD), mediated by Fas-FasL interactions, eliminates chronically activated T cells after an immune response to prevent autoimmunity. The extrinsic apoptosis pathway (caspase-8 activation at the DISC) is the execution mechanism for AICD.
Gene expression 17.05.02. AIRE-driven ectopic expression of tissue-specific antigens in mTECs is a remarkable example of transcriptional regulation: a single transcription factor activates thousands of genes that are normally silent in the thymus. The epigenetic mechanism involves AIRE-mediated recruitment of the transcriptional machinery to otherwise silent chromatin. V(D)J recombination is also regulated at the level of chromatin accessibility — RAG enzymes only access loci that are actively transcribed.
Evolutionary biology. The germinal center reaction is a microcosm of Darwinian evolution operating within an individual organism: mutation (AID), selection (competition for antigen and T cell help), and heritable variation (mutations passed to daughter cells). Affinity maturation over 2-3 weeks produces the same qualitative outcome as natural selection over millennia — improved fit between a receptor and its ligand — compressed into days.

Historical notes Master

The concept of adaptive immunity emerged from the observation that survivors of certain infectious diseases were protected from subsequent attacks. Thucydides described this phenomenon during the plague of Athens (430 BC), noting that the same person was never attacked twice. In medieval China and the Ottoman Empire, variolation (deliberate inoculation with smallpox material) was practised as a form of immunisation centuries before its mechanism was understood. Edward Jenner's demonstration in 1796 that cowpox inoculation protected against smallpox established the principle of vaccination, though the cellular and molecular basis remained unknown for over 150 years.

The cellular basis of immunity was revealed by Elie Metchnikoff's discovery of phagocytosis in 1882 and Paul Ehrlich's side-chain theory of antibody formation in 1897. Ehrlich proposed that cells produce "side-chains" (receptors) that bind toxins, and that binding stimulates production of more receptors that are released into the blood as antibodies. Though incorrect in detail, Ehrlich's theory anticipated the concept of cell-surface receptors and selective stimulation — ideas that would resurface in Burnet's clonal selection theory 60 years later.

The existence of two distinct lymphocyte populations was established in the 1960s. Jacques Miller demonstrated that thymectomy in neonatal mice abolished cell-mediated immunity (thymus-derived T cells), while Henry Claman and colleagues showed that antibody production required both bone-marrow-derived cells (B cells) and thymus-derived cells (T cells). Max Cooper and Robert Good established the B cell (bursa-equivalent, bone marrow) and T cell (thymus) lineages in chickens and extended the findings to human immunodeficiency diseases.

Macfarlane Burnet proposed the clonal selection theory in 1957 (formally published in his 1959 book The Clonal Selection Theory of Acquired Immunity). The theory resolved a fundamental puzzle: how could the body produce antibodies against synthetic molecules that had never existed in nature? The Darwinian answer was that the antibody repertoire is generated randomly before antigen encounter, and antigen selects the matching clone for expansion. David Talmage (1957) independently proposed a similar idea. The theory was confirmed experimentally by Gustav Nossal and Joshua Lederberg in 1958, who demonstrated that single B cells produce antibodies of only one specificity (single-cell analysis of individual antibody-forming cells), and by subsequent work showing that antigen binding triggers clonal proliferation.

The molecular mechanism of antibody diversity was solved in the 1970s and 1980s. Susumu Tonegawa demonstrated in 1976 that immunoglobulin genes undergo somatic rearrangement during B cell development — the V, D, and J gene segments are separate in the germline genome and are joined by DNA recombination in the B cell. This discovery (Nobel Prize, 1987) explained how a finite genome could encode an effectively infinite repertoire of antibodies. The subsequent identification of RAG1/RAG2 (Schlissel, Baltimore, and colleagues, 1989), TdT, and the NHEJ machinery completed the picture of V(D)J recombination.

Somatic hypermutation remained mechanistically mysterious until the discovery of AID by Tasuku Honjo's laboratory in 2000. Muramatsu and colleagues showed that AID expression was necessary and sufficient for both somatic hypermutation and class switch recombination, and that AID deficiency in humans causes Hyper-IgM syndrome type 2 (failure of class switching, resulting in high IgM and low IgG/IgA/IgE). The biochemistry of AID — a single enzyme that deaminates cytidine in DNA to initiate both processes — resolved a decades-long puzzle about how antibody diversification is achieved.

The discovery of T cell subsets and the MHC restriction phenomenon transformed immunology. Peter Doherty and Rolf Zinkernagel showed in 1974 that cytotoxic T cells recognise viral antigens only when presented by the host's own MHC molecules (MHC restriction, Nobel Prize, 1996). This finding required the concept of a T cell receptor that simultaneously engages both antigen and MHC — a receptor whose molecular identity would not be discovered until the 1980s. The TCR was cloned by Tak Mak and Mark Davis in 1984.

The two-signal model of T cell activation was articulated by Polly Matzinger (1994) in her "danger model," building on earlier work by Kevin Lafferty and Alistair Cunningham. Matzinger proposed that the immune system responds not to "non-self" per se but to danger signals from damaged or stressed cells. This framework explained why transplants between identical twins (non-self but not dangerous) are tolerated, and why tumors (self but dangerous in principle) are sometimes attacked. The molecular correlate of the danger model is the two-signal requirement: antigen (signal 1) plus innate immune-derived co-stimulation (signal 2, triggered by damage or infection).

Checkpoint immunotherapy began with James Allison's demonstration in 1996 that blocking CTLA-4 in mice could reject established tumors. Ipilimumab (anti-CTLA-4) was approved for melanoma in 2011. PD-1 was discovered by Tasuku Honjo in 1992, and its role as an inhibitory receptor was established over the following decade. Anti-PD-1 antibodies (nivolumab, pembrolizumab) were approved for melanoma and lung cancer in 2014-2015. The 2018 Nobel Prize to Allison and Honjo recognised the translation of basic immunological checkpoint biology into transformative cancer therapy.

Bibliography Master

Alberts, B. et al. — Molecular Biology of the Cell, 7th ed. (Garland Science, 2022), Ch. 24 The Adaptive Immune System.
Lodish, H. et al. — Molecular Cell Biology, 9th ed. (W. H. Freeman, 2021), Ch. 24 Immunology.
Murphy, K. & Weaver, C. — Janeway's Immunobiology, 10th ed. (Garland Science, 2022), Ch. 1-5.
Burnet, F. M. — The Clonal Selection Theory of Acquired Immunity (Cambridge University Press, 1959).
Tonegawa, S. — Somatic generation of immune diversity, Nobel Lecture, In Vitro Cellular & Developmental Biology 24 (1988) 105-115.
Muramatsu, M. et al. — Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme, Cell 102 (2000) 553-563.
Doherty, P. C. & Zinkernagel, R. M. — A biological role for the major histocompatibility antigens, Lancet 305 (1975) 1406-1409.
Matzinger, P. — Tolerance, danger, and the extended family, Annu. Rev. Immunol. 12 (1994) 991-1045.
Leach, D. R., Krummel, M. F. & Allison, J. P. — Enhancement of antitumor immunity by CTLA-4 blockade, Science 271 (1996) 1734-1736.
Iwai, Y. et al. — Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade, Proc. Natl. Acad. Sci. USA 99 (2002) 12293-12297.
Nossal, G. J. V. & Lederberg, J. — Antibody production by single cells, Nature 181 (1958) 1419-1420.
Miller, J. F. A. P. — Effect of neonatal thymectomy on the immunological responsiveness of the mouse, Proc. R. Soc. Lond. B 156 (1962) 415-428.
Cooper, M. D., Peterson, R. D. A., South, M. A. & Good, R. A. — The functions of the thymus system and the bursa system in the chicken, J. Exp. Med. 123 (1966) 75-102.
Mak, T. W. & Saunders, M. E. — The Immune Response: Basic and Clinical Principles (Elsevier, 2006).