Lecture 15: Tolerance and Immunoregulation

Aim of the Immune System

• To recognise and removal all danger

  • Want a targeted and regulated response to avoid self hard

Innate Immunity

• Address need for broad recognition – involves Pattern Recognition Receptors – recognise MAMPs/PAMPs and DAMPs

  • encoded in the Germline, I.e. Inherited and has evolved over many generations to generate broad recognition receptors

Adaptive Immunity

• Generates a highly diverse, random repertoire of BCRs (antibodies) and TCRs.

• Diversity is created through somatic recombination: rearrangement of multiple gene segments (V, D, J) in developing B and T cells to form a V-exon.

• This process is not dependent on knowing the specific threat—it aims to produce enough receptor types to eventually recognise any danger.

• Each lymphocyte is clonal: it expresses a single unique receptor.

• Broad receptor diversity makes it hard for microbes to evade detection.

Risk of Adaptive Immunity

• While broad recognition is beneficial, it carries the risk of autoimmunity.

• Some randomly generated receptors may target the body’s own tissues.

• The immune system must carefully manage this risk through tolerance mechanisms.

Mechanisms of Tolerance

• Two types

  • Limit production of dangerous self-reactive T and B cell clones → mechanics present in cell development

  • Prevent unwanted destructive responses by any clones produced → mechanism present to restrain the activity of self reactive cells that enter the peripheral circulation

Self-Reactivity

• Normal within a healthy immune system

  • But is restrained by mechanisms

Tolerance

• Failure to respond to intrinsic self-antigens (self) or external antigens

• It is antigen-specific

• Important to prevent reactions against:

  • harmless non-self-antigens, e.g. commensal bacteria; fetomaternal antigens

  • therapeutically relevant antigens, e.g. allergens; transplantation antigens mechanisms used therapeutically

Burnet and Medward Nobel Prize (1960)

• Awarded for their discovery of acquired immunological tolerance – work influenced by Ray's Owens

  o    Don’t inherit self-tolerance – it is acquirer

• Don’t inherit adaptive receptor; acquire repertoire      

• Development of f lymphocyte receptor through B-cell and T-cell development

• The body actively learns not to react to self during the development and selection of immune cells.

Evidence for the Acquisition of Self Tolerance - Owen & Burent) (1940’s)

• Observations that non-identical twin calves sharing a common placenta and blood supply became tolerant of each other's tissues.

  • Their blood systems showed chimerism—each had immune cells from the other.

  • A blood transfusion from one twin to a non-identical calf led to rejection, showing that tolerance was specific.

• Suggests that tolerance is induced to antigens shared as a fetus.

  • Proposed that the immune system tolerates antigens present during foetal development.

Evidence for the Acquisition of Self-Tolerance - Medawar (1953)

• Introduced donor cells in utero to foetal mice from a different purebred strain.

• As adults, these mice could accept skin grafts from the donor strain they were exposed to in the womb.

  • Donor cells delivered to a foetus mouse in utero allowed adult offspring to accept skin grafts from the same donor strain

• This showed the mice had developed specific tolerance to the donor antigens encountered in utero—i.e., they had acquired immunological tolerance.

Clonal Selection Theory

• States that there are relatively few lymphocytes with a different antigen receptor.

  • Each lymphocyte expresses a unique receptor.

• When a mature lymphocyte encounters an antigen that matches its receptor, it becomes activated.

• The activated cell then proliferates (clonal expansion) to produce many identical cells that help combat the antigen.

Clonal Deletion

• Process where immature or developing lymphocytes that bind strongly to an antigen during development are eliminated (die).

• It occurs in the thymus (T cells) and bone marrow (B cells) to help prevent autoimmunity.

• It was once thought to explain self-tolerance, but this is not the case—other mechanisms (like clonal anergy and regulatory T cells) also contribute

Compromise When Establishing Protection Against Autoimmunity

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

• Clonal deletion helps eliminate self-reactive lymphocytes, but it creates holes in the immune repertoire—blind spots that microbes could exploit.

• Therefore, establishing tolerance is more nuanced:

  • It involves multiple mechanisms beyond deletion.

  • These occur in a layered series of checkpoints, where the most dangerous cells are eliminated, and less dangerous ones are controlled or tempered (e.g., via regulatory T cells or anergy).→ helps to keep broad protections

• This balance ensures immune protection is maintained without triggering autoimmunity.

Mechanisms Contributing to Tolerance

• Inactivation or removal of potentially self-reactive lymphocyte clones:

  • cell death, receptor editing, or anergy.

• Immune regulation:

  • Modulates or tempers the response of potentially dangerous cells.

  • Involves suppression (e.g., by regulatory T cells) and functional deviation (redirecting responses).

• Limited access to self-epitopes:

  • Some antigens are not available for immune recognition due to the context of their expression

  • Includes ignorance, sequestration, and immune privilege (e.g., in the brain, eyes, or testes).

Central Tolerance

• Occurs in the primary lymphoid organs (bone marrow and thymus).

• It plays a central role in removing highly self-reactive clones during lymphocyte development.

  • Mechanisms include deletion, receptor editing, anergy, and functional skewing (towards tTreg).

• Central tolerance is not absolute—some self-reactive cells escape into the periphery.

• These mechanisms in the periphery limit or restrain reactivity to self or harmless antigens.

Peripheral Tolerance

• Occurs in secondary lymphoid organs (e.g., lymph nodes, spleen) and peripheral tissues.

• Multiple mechanisms help limit reactivity against self or harmless antigens in the periphery.

  • Mechanisms include:

    • Ignorance, anergy, deletion, functional skewing, and regulation (e.g., through regulatory T cells).

  • Lack of T cell help for B cells can also limit self-reactivity.

• Self-reactive B and T cells can enter the peripehry - these mechanisms are present, which act to contain this.

B-Cell Development

• occur in the bone marrow.

  1. The heavy chain gene undergoes rearrangement, starting with the D segment joining the J segment, followed by the V segment joining the DJ.

  2. Pre-B-cell proliferative stage: the first checkpoint, an antigen-independent quality checkpoint. It checks if the heavy chain gene rearrangements generate a chain that can associate with the surrogate light chain to form a functional surface receptor.

     • If rearrangement fails (no open reading frame or improper folding and can’t join with light chain), the cell is ineffective and will not become a defence cell.

     • If successful in reaching the surface, the heavy chain signals back to the cell (cross links with the surrogate light chain), temporarily downregulating RAG enzymes and triggering proliferation.

       • heavy chain loci no longer avalible for recombination - ensures some exclusion

  3. Once the heavy chain is successful, RAG genes are reactivated for light chain recombination. This creates a VJ recombination at the light chain loci, aiming for a functional receptor, following the generation of a light chain that can bind to the heavy chain and rech the surface.

  4. The first tolerance checkpoint checks if the immature B-cell binds to self-antigens in the bone marrow - determines the fate of the cell

     • If the cell binds self-antigens, it may undergo editing or deletion to prevent autoimmunity.

Factors Affecting B-Cell Fate

• Determined by their interaction with self-antigen in the bone marrow.

• Key factors influencing this interaction:

  • The concentration of the self-antigen.

  • The ability of the antigen to cross-link B-cell surface receptors.

• These factors influence whether the B-cell is allowed to mature, undergo receptor editing, become anergic, or is deleted to prevent self-reactivity.

What determines whether an immature B-cell in the bone marrow survives and matures?

• If an immature B-cell does not strongly react with self-antigen in the bone marrow, it continues to mature and migrates to the periphery, expressing both IgM and IgD on its surface.

• If the B-cell undergoes strong, multivalent cross-reactions with self-antigens:

  • This indicates potentially dangerous self-reactivity.

  • The cell is either:

    • Deleted via apoptosis, or

    • Rescued through receptor editing (to attempt a non-self-reactive receptor).

      • If sucessful - exits to the periphery

      • If unsuccessful - clonal deletion

Receptor Editing

• Allows the immature B-cell to have “another go” at generating a safe receptor if the initial one is self-reactive.

• It occurs due to the organisation of gene segments in the light chain locus (V and J segments).

• If a dangerous V exon is present and binds in a dangerous manner:

  • The RAG genes remain active, allowing further V–J recombination.

  • A new V and J segment can be joined on either side of the original one.

  • This gives the cell another chance to generate a non-self-reactive light chain and avoid deletion.

Fates of Immature B-Cells in the Bone Marrow Based on Interactiosn With Self-Antigens

• Soluble self-antigen binding (not enough cross-reactivity for deletion):
  → Cells migrate to the periphery and become anergic

  • Anergic cells:

    • Low IgM (retained intracellularly), normal IgD (surface)

    • Tend not to signal

    • Unresponsive and short-lived (can't compete for survival signals and rapidly die)

• Low-affinity self-binding (weak cross-reactivity interaction):
  → Cells are not anergic, but may still bind self-antigen

  • In periphery:

    • May not encounter antigen at sufficient level or in the right context

      • Signal not strong enoguh to cause deletion or anger

    • they remain ignorant - if they dont see antigen at high concentrations or in a sufficent context -not activated

    • Antigen may be sequestered in the periphery preventing activation and binding to cell

Summary of B-Cell Fates in Central Tolerance

• Self-reactive clones DELETED - or rescued by editing

• Self-reactive clones ANERGIC (unresponsive) – die rapidly

• Self-reactive, but IGNORANT

T-Cell Development in the Thymus

• Cells are derived from a common lymphoid progenitor in the bone marrow which enters the bloodstream to travel to the thymus

• In the thymus, T-cel interactions with the stroma to drive its development and commitment

• * 95% of cells derived form the thymus are alpha beta t-cells

Stages of T-Cell Development

• Based on the expression of co-receptor molecules (CD4/CD8)

  • Early Thymocyte

  • Double Negative (DN) Thymocytes → No CD4/CD8

  • Double Positive (DP) Thymocytes → Both CD4/CD8

  • Single Positive (SP) Thymocytes → Lineage commitment phase

    • Express either CD4 or CD8

TCR Antigen Recognition

• Recognise processed antigen presented as a peptide in the groove of MHC (I/II)

  • Receptor interacts with both MHC and the peptide

• The receptor interacts with the footprint of CDR1, 2, 3 from the alpha-beta chains – interacts with both TCR and peptide

Key Developmental Checkpoints in T-Cell Development

• TCR gene rearrangement can lead to different clonal outcomes:

  1. Recombination generates alpha and beta gene loci that fails to express surface TCR → USELESS (no antigen receptor = no function)

  2. TCR that can't recognise self-MHC → USELESS

     • Can't participate in antigen recognition or immune surveillance

  3. TCR recognises self-MHC/self-antigen too strongly → HARMFUL

     • Deleted during development (autoimmune risk)

     • Checkpoints in development remove these T-cell clones

  4. TCR recognises self-MHC, but not self-antigen too strongly → USEFUL

     • Survive and mature; ready to detect antigens in the periphery

• Thymic selection is highly stringent:

  • Only ~2% of cells with a functional TCR survive the thymic selection and enter the periphery

TCR Development Checkpoint 1: Quality Check

• Recombination at the TCR β chain locus

• First quality checkpoint:

  • Does the TCR β chain successfully pair with a surrogate α chain to form a pre-TCR?

  • If yes → generates a survival signal:

    • Downregulates RAG

    • Triggers proliferation

    • Begins TCR α chain rearrangement

• If no survival signal → cell undergoes apoptosis

• Key question at this checkpoint:

  • Has TCR β gene rearrangement resulted in functional pre-TCR expression?

T-Cell Development in the Thymus

• Different regions of the thymus support different checkpoints in T-cell development

  • Facillitates different selection criteria

• Progenitor cells enter thymus near the cortex-medulla boundary → migrate outward into the cortex

• In the cortex:

  • Cells at the Double Negative (DN) stage undergo an antigen-independent quality check

• Cells then move deeper into the cortex, moving to the centre, becoming Double Positive (DP) thymocytes

  • Undergo positive selection via interaction with MHC + antigen peptide expresseed on cortical epithelial cells

• Surviving DP thymocytes migrate to the medulla

  • Face the next checkpoint involving interactions with medullary epithelial cells and dendritic cells (central tolerance)

• Regional movement through thymus enables distinct checkpoints to interrogate different features of TCR specificity and self-reactivity

T-Cell Development Checkpoint 2: Positive Selection of T Cells

• Key Question: Can the rearranged αβTCR recognise self-MHC?

• Interaction between double-positive (DP) thymocytes and cortical epithelial cells in the thymus

• If recognition of self MHC and self-antigen occurs:

  • Thymocytes receive a survival signal

  • Cells become single positive (SP) (CD4⁺ or CD8⁺)

  • Migrate to the medulla for the next selection phase

• If recognition fails:

  • RAG genes remain active, allowing further α-chain recombination (secondary V-J joining)

  • If a new functional α-chain is produced and recognises self-MHC → cell survives and progresses as CD4/CD8 cell

  • If not → cell fails to receive signals and dies after 3–4 days

T-Cell Development Checkpoint 3: Negative Selection of T Cells

• Key Question: Does the αβTCR recognize self-MHC + self-peptide too strongly?

• Process occurs in the medullary region of the thymus

  • T cells increase speed of movement to scan MHC peptides on dendritic cells (DCs) and medullary epithelial cells

• Aims to eliminate T-cell clones that bind too strongly to self-antigen — harmful and deleted

• Challenge: Need to screen T cells against

  • Thymic antigens

  • Tissue-specific antigens not normally expressed in the thymus

AIRE and FezF2

• Transcription regulators expressed by medullary epithelia

• They allow for promiscuous gene expression

• Results in a mosaic of peptides expressed that would not normally be present in the thymus → represents peptides from various tissues to help inform negative selection

Affinity Hypothesis of T-Cell Selection

• Idea that T-cell fate in development is determined by TCR binding strength (affinity) to self-MHC + self-peptide and its signalling strength

Affinity Hypothesis of T-Cell Selection: Weak Signalling/ Binding

• If the cell does not bind TCR→, it fails positive selection
  → T cell is useless and dies (can’t recognise MHC)

Affinity Hypothesis of T-Cell Selection: Too Strong Binding

• Triggers negative selection
  → T cell is potentially harmful and deleted

Affinity Hypothesis of T-Cell Selection: Intermediate Binding

• T cell passes selection, surives and enters the periphery

  • Capable of contributing to immune defence

Affinity Hypothesis of T-Cell Selection: Moderate to Strong Binding

• T cell becomes functionally skewed
  → Differentiates into thymic-derived regulatory T cells (tTregs) that express Foxp3 and function in the periphery to regulate self-reactivity

Thymic Emigrants

• 2% of cells that pass through thymic selection form functional receptors

  • CD8 cytotoxic cells

  • CD4 helper cells (can be skewed into various effector functions upon activation)

• 2-3% of CD4 cells leave the thymus as Foxp3+ Tregs (regulatory T cells)

Activation of T-Cells

• Signal 1 = antigen-specific activation signal (TCR/MHC:pep)

• Signal 2 = co-stimulus

• Signal 3 = cytokines (differentiation)

  • Integration of these signals determines the type of T-cell function

• Activation required by B-cells to mount an antibody response

  • no T-cells = no B-cell response

Mechanisms of Peripheral Tolerance in B-Cells

• Activation of PPRs on DCs will result in phagocytosis of antigens and display them on their surface.It will also upregulate its co-stimulatory molecules and alter its migratory properties, moving to the draining lymph node and to the T-cell area, where T-cells scan for the antigen.

  • If T-cells recognise the antigen with co-stimulation and cytokines, they are activated.

• Foreign antigens can drain the lymph node and be recognised by BCRs in the B-cell area

  • Antigen is processed and presented on the B-cell surface.

• B-cells and activated T-cells move to the B-T cell boundary.

  • T-cells recognise foreign antigens on MHC, get activated, and provide co-stimulation and cytokines to activate the B-cell to produce plasma cell antibodies.

Peripheral Tolerance in B-Cells & Lack of T-Cell Help

• Self-antigens may bind to and be taken up B-cells where it is processed.

• When B-cells look for T-cell help, there is no activation assistance for self-antigens.

• Main Mechanism of Peripheral Tolerance:

  • The lack of T-cell help

• Within germinal centres, B-cells must compete for antigen from follicular DCs.

• T-follicular helper cells are present, but no help/signaling occurs in response to self-antigen.

Signalling from Dendritic Cells Determines T-Cell Activation & Tolerance

• Antigen must be presented by DCs with co-stimulation to activate naïve T-cells.

• If co-stimulation is absent (antigen presentation without danger), it leads to T-cell inactivation or tolerance.

  • Results in anergy or regulation of T-cells.

Clonal Anergy → Turning T-Cells Off

• SIGNAL 1 ALONE renders a T cells unresponsive to antigen

  • e.g. Antigen encountered in the absence of DANGER

• PRRs form the innate immune system can act to recognise danger and is communicated  to the adaptive system

Regulation: Peripheral Induced Treg (pTreg)

• Recognition of antigen with an immunosuppressive signal 3 (cytokines), T-cells can be driven to become (peripheral) Tregs

Thymic-Derived Tregs (tTreg) vs. Induced Peripheral Tregs (pTreg)

• Thymic-Derived Tregs (tTreg):

  • Generated in the thymus.

  • FOXP3 positive, recognising self-antigen.

• Induced Peripheral Tregs (pTreg):

  • Can be FOXP3+/-.

  • Recognise self-antigen or mediate tolerance against harmless, non-self antigens (e.g., gut microbiota).

  • Help in tempering immune responses appropriately.

Regulation by Tregs

• Act to inhibit T-cells in many ways (still not fully understood) e.g.

  • Production of anti-inflammatory, pro-regulatory cytokines; TGFβ, IL10, IL35 (dependent on signals driving differentiation of the Treg)

  • Modulation of dendritic cells – become tolerogenic – drives DC tolerance not activation

  • Outcompete effector T cells for resources; IL2 (Treg commonly express IL2R)

  • Direct killing of T cells

  • Exosome transfer of miRNAs

Gut Immunology - Tolerogenic Environment

• Not all tissues are immunologically equivalent – different areas of antigen encounter may have different requirements for the immune system

• The gut has a thorough barrier that absorbs nutrients while being exposed to food antigens, bacteria, and other elements.

• The immune system in the gut is tuned to be more tolerogenic to prevent constant inflammatory reactions

Oral Tolerance

• The immune system's ability to develop tolerance to antigens ingested through the tolerogenic environment of the gut, rather than mounting an immune response.

• Historically, American Indians ingested small amounts of poison ivy leaves to prevent allergic reactions (Dakin, 1829).

• Eating an antigen for the first time in the tolerogenic environment of the gut helps the immune system tolerate it.

• Desensitisation therapies use this principle, giving small amounts of an allergen to temper the immune response over time against it

Immune Privilege Sites

• Specialised areas of the body that have eveoled to protect vital organs(e.g., brain, eye)

• If an antigen is found here a destructive immune respons is not intiated

• Grafts placed in these sites are not rejected

Characteristics of Immune Privilege Sites

• Enclosed physical barriers (limited lymphatic drainage and selective entry)

• Low MHC I expression (reduces immune activation and response)

• Rich in suppressive cytokines (e.g., TGFβ)

• FasL expression (triggers T-cell apoptosis if response is activated)

• Grafts in these sites are not rejected.

• Antigens in these sites don’t elicit destructive immune responses but can be targets for autoimmune diseases, if not subject to peripheral tolerance mechanisms

Sympathetic Ophthalmia

• Injury to one eye can cause the release of antigens in an inflammatory situation, which can drain into the lymph node

• Ignorant T-cells (that were previously unexposed to these antigens) now encounter these antigens in an inflammatory context.

  • These T-cells then drain back to both the injured and healthy eye, causing damage to both eyes

• The immune response is not tempered by peripheral tolerance to these antigens, as the antigen was not initially encountered in a controlled (non-inflammatory) environment.

  • peripheral tolerance not informed in advance

Immune Suppresion in Tumours

• Tumours can adapt to generate immune suppressive areas, effectively creating an immune privilege zone.

  • This immune suppression allows tumours to evade immune detection and attack.

• This must be overpowered by immunotherapies to overcome the immunosuppression to have an effect

Basis of Checkpoint Blockage as A Treatment of Cancer

• Antibodies interfere with the negative signal → take the brakes off" the immune system, enabling it to fight and provide protection against the cancer effectively

  • Pembrolizumab (Keytruda) and ipilimumab (Yervoy).

Inappropriate Immunity

• Lead to diseases like allergies and autoimmunity.

  • Can cause difficulties in tumour/ cancer treatments