Anergy and Tolerogenic Dendritic Cells in Peripheral Tolerance

Peripheral Tolerance and the Mechanism of Anergy

Conceptual Overview * Anergy is categorized as the third major mechanism or "failsafe" of peripheral tolerance. * The lecture outcome focuses on appraising the importance of anergy and the role of tolerogenic dendritic cells in its generation.
Historical Context: Adjuvants and Danger Signals * Immunologists have indirectly observed the effects of anergy for decades, specifically through the study of adjuvants in the 1940s and 1950s. * Early Vacuum Experiments: Research demonstrated that injecting an animal with a protein alone (a vaccine) would fail to generate an immune response. * Known Mechanism: We now understand this failure is due to a lack of "danger signals" in the antigen-presenting cells (APCs). Specifically, there is an absence of costimulatory molecules $CD80$ and $CD86$ . * Consequence: The absence of these molecules does more than fail to activate the T cell; it actively turns the T cell off, inducing a state of anergy. * The Role of Adjuvants: To be effective, a vaccine requires an adjuvant containing Pathogen-Associated Molecular Patterns ( $PAMPs$ ) or Damage-Associated Molecular Patterns ( $DAMPs$ ). These signals activate a dendritic cell (DC), making it immunogenic rather than tolerogenic by inducing the expression of $CD80$ and $CD86$ .

Definition and Molecular Basis of Anergy

Defining Anergy * Anergy is the inability of an antigen-specific cell to respond to a subsequent antigenic challenge. * Classic Explanation: Anergy results from antigen presentation occurring in the absolute absence of costimulation.
Comparison of States * Homeostasis: In the absence of $PAMPs$ or $DAMPs$ , the dendritic cell is "tolerogenic." It lacks $CD80$ and $CD86$ expression. When it presents antigen to a T cell, the result is anergy rather than activation. * Infection/Inflammation: The presence of $PAMPs$ or $DAMPs$ causes the DC to express $CD80$ and $CD86$ . This allows the DC to transduce "signal two" to the T cell, triggering the production of Interleukin-2 ( $IL-2$ ) and subsequent T cell activation.
Molecular Mechanisms of T Cell Anergy * Interactions with a DC lacking costimulatory signals induce specific molecular changes in the T cell. * Ubiquitin Ligases: Anergic T cells upregulate ubiquitin ligases, primary among them being $GRAIL$ and $CBL-B$ . * Function of Ubiquitin Ligases: These molecules add ubiquitin (a small protein) to specific target molecules, marking them for degradation within the proteasome. * Targeted Molecules: - $GRAIL$ : Specifically targets $CD3\text{ }\text{zeta}$ ( $CD3\text{ }\text{ζ}$ ), which contains three Immunoreceptor Tyrosine-based Activation Motifs ( $ITAMs$ ) and is critical for signaling. - $CBL-B$ : Targets downstream signaling molecules including Phospholipase C gamma 1 ( $PLCγ1$ ) and Protein Kinase C theta ( $PKCθ$ ). * Result: The downregulation of $CD3\text{ }\text{ζ}$ , $PLCγ1$ , and $PKCθ$ prevents the T cell from signaling appropriately, rendering it nonresponsive. * Autoimmune Implications: Evidence for the importance of anergy is seen in mice deficient in $GRAIL$ or $CBL-B$ , which develop autoimmune diseases.

Anergy in B Cells

Conditions for B Cell Anergy * Anergy is not unique to T cells. B cells become anergic under two primary conditions: 1. Weak binding to self-antigen during development with minimal cross-linking. 2. Lack of T cell help in the periphery.
Phenotypic Identification * Anergic B cells undergo distinct surface protein changes. * $IgM$ vs. $IgD$ : The naive B cell downregulates surface $IgM$ while retaining surface $IgD$ . * Detection: An anergic B cell is identified as $IgD^{pos}$ and $IgM^{low}$ . The $IgM$ is retained within the cell rather than reaching the surface. * Function of $IgD$ : The exact role of $IgD$ is not fully understood, but it appears to be vital in maintaining tolerance.
Signaling Blockage * Similar to T cells, anergic B cells downregulate signaling capabilities: - Blockage of $CD79$ ( $Ig\text{ }\text{α}$ and $Ig\text{ }\text{β}$ ). - Blocked phosphorylation of $SYK$ . - Blocked $NF-ζB$ signaling.

Generation of Tolerogenic Dendritic Cells

Anergic T cells are generated by interacting with non-stimulatory, tolerogenic DCs. The state of the DC is determined by several factors.
Mechanism 1: Absence of Danger Signals * Under homeostatic conditions (no infection or inflammation), Pattern Recognition Receptor ( $PRR$ ) signaling is absent. * Without $PRR$ signaling, $CD80$ and $CD86$ are not upregulated. * Self-antigens are presented in the absence of costimulation, leading to tolerance. * Non-professional APCs: Epithelial cells express Class I $MHC$ but not costimulatory molecules. Theoretically, naive T cells interacting with epithelial cells would become anergic. However, this is limited in vivo because naive T cells primarily recirculate through peripheral lymph nodes rather than peripheral tissues like epithelium.
Mechanism 2: Dendritic Cell Lineage Specialization * Some argue specific lineages are specialized for tolerance. * Langerhans Cells (LCs): Found in the epidermis of the skin (while dermal DCs occupy the dermis). * Specialization Hypothesis: LCs pick up antigen and migrate to lymph nodes. Some immunologists suggest they are specialized for tolerance, secreting $TGF-beta$ ( $TGFβ$ ) to promote regulatory T cells ( $Tregs$ ) and induce anergy. * Rationale: Antigens in the epidermis often include commensal bacteria or self-antigens, necessitating tolerance. Antigens penetrating the dermis imply damage or aggressive pathogens, requiring an immunogenic response. * Plasmacytoid DCs (pDCs): Known for producing Type 1 Interferon in antiviral immunity, they are also poor APCs. Some argue they induce tolerance, though the speaker expresses skepticism, noting that any poor APC might appear to induce tolerance in experimental settings.
Mechanism 3: Active Tolerogenic Signaling and Plasticity * Dendritic cells exhibit plasticity and can be actively directed toward a tolerogenic state via signaling. * Inflammatory/Immunogenic Pathway: Pathogens ( $PAMPs$ ) and Necrosis ( $DAMPs$ ) trigger $PRRs$ and the $NF-ζB$ pathway, leading to pro-inflammatory cytokines and upregulation of $MHC$ , $CD40$ , $CD80$ , and $CD86$ . * Tolerogenic Pathway: Abundant self-antigen is associated with apoptosis (programmed, non-inflammatory cell death) rather than necrosis. * $MERTK$ Receptor: DCs possess receptors for apoptotic material, such as $MERTK$ . * $SOCS$ Proteins: $MERTK$ signaling induces the expression of $SOCS1$ and $SOCS3$ (Suppressor of Cytokine Signaling). These molecules inhibit $PRR$ signaling and the $NF-ζB$ pathway, preventing the upregulation of costimulatory molecules and cytokines.

Oral Tolerance and the Gut Environment

Definition: Oral tolerance is the systemic non-responsiveness to antigens (like food) that have been ingested.
Experimental Evidence: Feeding an animal an antigen (e.g., ovalbumin) and later challenging it with an injection of the same antigen results in a poor immune response compared to control groups.
Cellular Mechanism: The gut contains specialized $CD103^{pos}$ dendritic cells.
Immunosuppressive Local Environment: These DCs are exposed to signals that maintain their tolerogenic state: - Retinoic Acid: A derivative of Vitamin A. - Cytokines: $TGFβ$ and $TSLP$ . - Short-chain fatty acids.
Outcome: These signals ensure DCs generate $Tregs$ and maintain tolerance to food antigens.

The Rationale for Anergy: Clonal Redemption

The Evolutionary Dilemma: Why keep anergic cells instead of deleting them via Activation Induced Cell Death ( $AICD$ )? Keeping autoreactive clones seems dangerous.
The Repertoire Gap Hypothesis: Deleting clones might create "holes" in the immune repertoire that pathogens could exploit.
B Cell Clonal Redemption: - Anergic B cells can be "redeemed" in a germinal center reaction if they encounter a very powerful antigenic stimulus and high-density cognate T cell help. - During the reaction, they undergo Somatic Hypermutation. - Outcomes: 1. If they remain self-reactive, they die by apoptosis. 2. If they mutate away from self-reactivity but retain/gain affinity for a foreign pathogen, they emerge as matured, useful clones.
T Cell Reactivation: - Experimentally, anergic T cells can be reawakened using an excess of $IL-2$ or by transfer into lymphopenic environments (driven by $IL-7$ and $IL-15$ ). - Uncertainty: It is unclear how often this occurs in vivo. Because T cells do not undergo somatic hypermutation, they remain self-reactive upon reawakening. - Proposed Utility: Some anergic T cells may serve as precursors for $Tregs$ , which would then help dampen autoreactivity.

Questions & Discussion

The AI Challenge: The speaker presented an AI-generated image illustrating DC/T cell interaction and stated, "There’s something wrong with this picture… AI stuffed it up." * Prompt: Students are challenged to identify the molecular immunology error in the image and post their findings on Moodle. * Warning: The speaker cautioned, "Always don't turn your brain off when you're using AI."
Skepticism on pDC Role: The speaker noted skepticism regarding whether plasmacytoid DCs have a specialized role in tolerance in vivo, suggesting their performance as poor APCs in experiments might mistakenly be interpreted as a specialized tolerogenic function.