Adaptive recognition and tolerance

What is the difference between innate and adaptive recognition?

• Innate immunity recognises PAMPs and DAMPs with germline-encoded PRRs.

• Adaptive immunity recognises a highly diverse, random repertoire of antigens with BCRs and TCRs, which are somatically generated by rearranging gene segments in developing B and T cells.

What are the benefits of adaptive recognition?

Adaptive immunity gives broad recognition, needs no prior exposure, provides memory, and is hard for pathogens to avoid. The downside is the potential for autoimmunity.

What are the principles of tolerance?

• Self-tolerance is acquired, not inherited.

• The immune system must remove dangerous self-reactivity without impairing broad recognition and effective defence.

• Tolerance is antigen specific. It can develop to intrinsic self antigens and also to external antigens such as harmless self antigens (fetomaternal) and therapeutic ones (transplant). Clonal deletion alone would leave "blind spots".

What are tolerance checkpoints?

• Tolerance uses multiple layers of checkpoints: delete the most dangerous reactivities, then control and tune down others to keep the breadth of recognition.

–    Limit production of self-reactive T and B clones during development.

–    Prevent unwanted destructive responses by any clones that enter the circulating pool.

What is central and peripheral tolerance?

• Central tolerance (primary lymphoid organs: bone marrow for B cells, thymus for T cells): removal of highly self-reactive clones during development, mainly by deletion and conversion to tTreg. Not absolute.

• Peripheral tolerance (peripheral organs, tissues, secondary lymphoid organs): tTreg suppression, ignorance, deletion, anergy, iTreg induction, and lack of T cell help for B cells.

What is the structure of a TCR/BCR?

• Immunoglobulin folds: the antigen-recognition portions of the BCR and TCR are structurally very similar. The variable region has 3 regions of hypervariability, which fold into 3D immunoglobulin folds. Bringing 2 immunoglobulin folds together forms a β-barrel that holds the 3 hypervariable domains to form the antigen-binding site.

• In the 3D structure, the variable regions' underlying framework can't tolerate much variability (it would disrupt the structure), whereas the 3 hypervariable loops can. The loops are complementarity-determining regions (CDR1–3).

How are the Fab regions different between the BCR and TCR?

The Fab region of the BCR and the TCR share structural similarities (immunoglobulin folds) but bind differently: the BCR binds native antigen; the TCR binds MHC-processed antigen. Once activated, the BCR becomes a soluble Ab whose Fc region recruits effector functions, whereas T cells act locally by cell-cell contact or local cytokine secretion.

What is the structure of a TCR?

• The TCR recognises peptide:MHC (self-restriction). CD4/CD8 bind invariant sites on MHC-II/MHC-I (lineage commitment).

• The TCR has variable, constant and hinge regions before the transmembrane region and cytoplasmic tail, and is associated with CD3.

How are TCR genes rearranged?

• α chain: the V segment joins J to form the V exon. V gives CDR1 and 2; V and J together give CDR3.

• β chain: 2 rearrangements: D joins J (DJ), then V joins DJ. V, D and J all contribute to CDR3.

How does diversity differ between the BCR and TCR?

BCRs and TCRs have similar combinatorial diversity, but the TCR has more junctional diversity: D segments are read in all reading frames, there are more J segments, and TdT acts at all junctions. Junctional diversity is concentrated in CDR3, which contacts the peptide the most.

What are gamma and delta genes?

γ and δ loci: there are 2 additional TCR germline loci. The γ locus is similar to the α locus (V and J segments); the δ chain has V, D and J (like β). The δ locus sits inside the α locus, with δ V segments interspersed among the α V segments, so joining an α V to a J completely removes the δ locus from being transcribed.

How does gene rearrangement machinery work?

• In germline DNA the constant region is an exon, but the variable region is made of V and J gene segments (not exons). As a progenitor commits to B or T lineage, lineage-specific, developmentally regulated machinery switches on, with epigenetic changes that loosen the DNA. A V segment joins a J segment to form a V exon; after transcription the intron between V and C is spliced out.

• RSS: each segment is flanked by a recombination signal sequence (conserved heptamer, spacer, conserved nonamer). The spacer is 12 or 23 bp, and the 12/23 rule says a 12 pairs only with a 23.

• RAG1/RAG2 (with HMG1) recognise the RSS, pull the segments together and cleave at the heptamer, leaving hairpin coding ends. The excised DNA in between circularises and is lost. Ubiquitous DNA repair machinery nicks the hairpins (making palindromic P nucleotides) and joins the ends imprecisely. TdT adds random N nucleotides.

What is a non-productive joint?

Imprecise joining can cause frameshifts or stop codons: a non-productive joint.

What are BCR checkpoints?

Checkpoints enforce clonality (allelic and isotypic exclusion): only one H and one L allele is used. Ig heavy chain rearranges first (D-J, then V-DJ), then light chain (κ first, then λ). A successful heavy chain pairs with a surrogate light chain at the first checkpoint, signalling proliferation and a halt to further heavy-chain rearrangement.

What is the double negative stage of T cell development?

• Double negative (DN): no CD4 or CD8; 4 stages.

–    DN1 and DN2: precursors commit to the T lineage; TCR loci still unrearranged.

–    DN3: β chain rearrangement begins (D→J, then V→DJ).

–    DN4: the TCR β chain pairs with the invariant pre-Tα to form the pre-TCR. Successful pre-TCR signalling halts further β rearrangement (allelic exclusion) and the cell proliferates rapidly.

What is the double positive stage of T cell development?

• Double positive (DP): proliferation stops; CD4 and CD8 are upregulated; α chain V→J joining. The α chain pairs with the existing β to make the mature αβ TCR.

–    Late DP: once a functional TCR can recognise MHC, RAG is switched off. DP cells die within 3–4 days if they don't bind MHC ("death by neglect"). Possible rescue by receptor editing (rearranging the other α locus).

What is the single positive stage of T cell development?

Single positive (SP): after positive selection. DP cells whose αβ TCR binds MHC-I better downregulate CD4 and keep CD8, and vice versa.

What are the checkpoints in T cell development?

• RAG-1/2: first active in β rearrangement (DN2–3); off after success (DN4); back on for α rearrangement (DP); permanently off after a successful αβ TCR.

• TdT: high through β rearrangement; falls during α rearrangement (mature α chains have far fewer N additions).

• Thymic education: after rearrangement, positive selection tests whether the receptor binds MHC, then negative selection induces apoptosis in cells with high self-affinity. How the receptor binds decides maturation on leaving the thymus. In peripheral tissues, cells gain functional competence.

• γδ T cells don't go through thymic selection like αβ cells.

• DN cells rearrange γ, δ and β genes at the same time, with quality checkpoints. A functional β chain usually outcompetes γ+δ rearrangement.

• Signals through the γδ TCR shut off β rearrangement. These cells leave as mature DN T cells (~5%) and go to epithelial sites. They are non-MHC restricted (innate-like), can secrete cytokines from the start, and give first-line defence.

• Strong ERK signalling → γδ; weak ERK signalling → αβ. The two cross-inhibit.

Where does each stage of T cell development occur?

The steps in T cell development don't happen in one place: cells move between thymic niches that provide the signals for each stage. This movement is driven by expression of different chemokine receptors as the T cells develop. The CLP enters the thymus from the blood near the cortico-medullary junction.

• DN1: medulla; DN2–4: outwards in the cortex towards the subcapsular region. After DN3, cells migrate back towards the medulla.

• Quality check (DN3): subcapsular region.

• Positive selection: near the cortico-medullary junction, with immature DP thymocytes and cortical epithelial cells (which detect the strength of antigen binding).

• Survivors enter the medulla for negative selection, which checks interactions between mature SP thymocytes and medullary epithelial cells and DCs.

How does antigen presentation differ in the cortex and medulla?

• Cortex: cortical thymic epithelial cells (cTECs) have constitutive MHC-I and II. MHC-I peptides: the thymoproteasome. MHC-II peptides: cathepsin L, thymus-specific serine protease (TSSP), constitutive macroautophagy. No costimulation.

• Medulla: DCs and mTECs have constitutive MHC-I and II. MHC-I peptides: housekeeping proteasomes and immunoproteasomes. MHC-II peptides: cathepsin S, macroautophagy. There is costimulation.

• Same MHC molecules, different peptide repertoire.

How do proteasomes differ in APCs?

• cTECs uniquely express the proteasome subunit β5t (thymoproteasome), which cleaves self proteins differently from any other cell: "private" peptides that bind MHC with low affinity. This weak interaction gives a gentle survival signal to immature thymocytes; a T cell that weakly recognises these cortical complexes survives positive selection.

• mTECs and DCs use the standard immunoproteasome; they use AIRE to express molecules normally found only in peripheral tissues: "public" peptides. High-affinity binding to a public peptide–MHC → clonal deletion or diversion into the Treg lineage. Moderate binding → exit to the periphery.

• By using private peptides in the cortex, the immune system coaxes T cells into recognising host MHC without exposing them early to the public self peptides they must learn to tolerate in the medulla.

How do mTECs present self-antigens?

• mTECs can express genes promiscuously, so they present tissue-restricted antigens (TRAs).

• mTECs are inefficient at taking up and presenting exogenous antigen. They express AIRE, which interacts with many transcription-related proteins and promotes lengthening of "stalled" transcripts, and Fezf2, a zinc-finger TF with an AIRE-independent, non-redundant role. Together they allow promiscuous expression of TRAs not normally made in the thymus. These antigens are processed and loaded on MHC-I.

• These endogenously expressed TRAs also reach MHC-II through macroautophagy, since mTECs are poor at taking up exogenous antigen, so they can be presented to CD4⁺ thymocytes as well as CD8⁺ ones.

What other sources of self-antigen are there?

• DC subsets sample antigen from mTECs (shed TRAs and mTEC debris) and from the periphery via the blood, and can cross-present ingested antigens.

• Immigrant DCs from the periphery carry peripheral self antigens (and harmless foreign ones) into the thymus via the blood and present them to developing thymocytes. This extends negative selection and tTreg induction to antigens made outside the thymus.

Why is promiscuous gene expression important?

Mosaic patterns of promiscuous gene expression (driven by AIRE and Fezf2) and mimetic cells (expressing genes driven by specific TFs) ensure negative selection in the medulla can tolerise against antigens normally made outside the thymus.

What is the affinity model?

Affinity model: clones that bind nothing die by neglect; clones that bind too strongly are deleted. Intermediate affinity survives and exits (2–5% of DP thymocytes). Cells with high affinity (not as high as the deleted ones) can exit as Foxp3⁺ tTregs (CD4 only).

How do T cells decide to express CD4 or CD8?

• Lineage commitment: MHC recognition on cTECs (cTEC peptidome) decides CD4 vs CD8 and function. Commitment requires silencing one co-receptor and starting a helper programme (cytokines) or a cytotoxic programme (killing genes).

• Kinetic signalling model: DP cells first lower CD4 and CD8, then re-express one, leaving a DP cell with lower expression of one co-receptor. The default path is CD8 signalling → Runx3, which silences CD4. Continuous CD4 expression → ThPOK, which inhibits Runx3. The cell is now SP.

What are recent thymic emigrants?

• SP thymocytes mature further before exit. They leave via blood vessels (not lymphatics), with S1P1 upregulation driving exit towards high S1P in blood.

• Recent thymic emigrants (RTEs) are less mature than circulating naive T cells (they don't proliferate or secrete cytokines on stimulation). They express CD62L and CCR7 → home to peripheral lymphoid organs and mature further there, with more tolerance checks as they meet peripheral antigens.

• RTEs gain cytokine production with epigenetic changes at cytokine loci. Their inability to make Th1 cytokines may promote peripheral tolerance: CD4⁺ RTEs convert to iTregs more readily than mature naive cells. RTEs that meet enough self antigen for the first time in the periphery are eliminated (activation-induced cell death, ACID) or made anergic.

• As RTEs mature they improve survival: IL-7 is needed to maintain Bcl-2, and IL-7Rα (induced in positive selection) keeps rising, as they join the long-lived naive pool.

How do DCs induce tolerance?

Tolerogenic DC signalling: presenting antigen without costimulation (no danger) is tolerogenic; signal 1 alone makes the T cell anergic. An immunosuppressive signal 3 (cytokines) drives peripheral Tregs. tTregs recognise self antigen; pTregs recognise self and harmless non-self antigens.

What is B cell peripheral tolerance?

B cell peripheral tolerance is mainly lack of T cell help: a B cell may present self antigen but no T cell recognises it.

What are immune privileged sites?

Immune privilege sites (brain, eye, etc.) are enclosed by physical barriers (limited lymphatic drainage, selective entry), have low MHC-I, are rich in suppressive cytokines and express FasL (killing responding T cells). They can still become autoimmune targets (e.g. sympathetic ophthalmia: eye injury releases antigen to nodes, and effector T cells then attack both eyes). Tumours can recruit immune suppression to gain privilege.