T Cell Development, Antigen Recognition, and Effector Functions — Detailed Study Notes

The Connection Between Infection Type and T Cell Activation

• Goal: Understand how the nature of an infection drives the cascade that activates the right T lymphocytes with specialized functions to defend the host against that infection.
• Emphasis on the link between the pathogen’s location/type (intracellular vs extracellular) and the T cell response needed.

T Lymphocyte Subsets and Their Specialized Roles

• Five distinct T cell subsets with specialized functions to defend against particular infections:
  • CD8+ cytotoxic T lymphocytes (CTLs)
  • CD4+ TH1 cells
  • CD4+ TH17 cells
  • CD4+ TH2 cells
  • CD4+ TFH cells
• (Note: There are other CD4+ cells not listed as main subsets here.)

Antigen Processing and Presentation as Central Activator

• Activation of T cells hinges on processing and presentation of protein antigens by Antigen Presenting Cells (APCs).
• The type of antigen presentation is directly linked to the resulting immune defense.
• Key idea: Antigen processing/presentation determines which T cell subset is activated and what effector response is elicited.

Antigen-Presenting Cells (APCs) and T Cell Activation

• APCs initiate most T cell responses.
• Main APC types:
  • Dendritic cells
  • Macrophages
  • B lymphocytes (B cells)
• Process: Antigen X is presented to T cells via the APC–T cell synapse; cytokines from APCs shape T cell differentiation.
• Outcome: Activation of T cells toward the appropriate effector function for the infection type.

Pathogen Localization and T Cell Strategy

• Pathogens can reside in three main locations:
  1) Intracellular cytoplasmic
  2) Intracellular intravesicular
  3) Extracellular (pathogens grow outside host cells and rely on host cell machinery/antibody responses）
• To defend successfully, specific T lymphocyte subsets must be activated to direct immune responses that reach the pathogen, regardless of whether it’s intracellular or extracellular.

CD8 T Cells: Role in Immune Defenses

• Focus on cytotoxic CD8+ T cells (CTLs) as a major component of defense against intracellular pathogens.
• Core idea: CTLs recognize antigens presented by MHC class I on infected cells and kill those cells to remove the pathogen’s niche.

Intracellular Infection: Antigen Source and CTL Challenge

• Intracellular infection definition: pathogens that live in a host cell’s cytoplasm and rely on host biosynthetic machinery.
• Result: Microbial antigens appear as peptides in the host cell cytoplasm.
• CTLs provide defense by killing the host cells that harbor intracellular pathogens (classic example: viruses; also some intracellular bacteria).
• Challenge for the immune system: CTLs must distinguish infected host cells from uninfected ones.

Strategy of CTL-Mediated Defense in Intracellular Infection

• CTLs must process antigen so that it:
  • activates CTLs, and
  • is displayed as a surface marker on infected cells for CTL targeting.

Endogenous Antigen Processing Pathway (for Cytosolic Antigens)

• Microbial antigens in the host cytoplasm are tagged with ubiquitin, signaling uptake and processing by proteasomes.
• Key question: What determines the fate of cytoplasm-produced protein antigens?
• Pathway components:
  • Endoplasmic reticulum (ER)
  • Cytosol
  • Proteasome
• The endogenous pathway leads to generation of peptide fragments that will be loaded onto MHC class I molecules for surface display.

Endoplasmic Reticulum and Cytosol in Antigen Processing

• Proteasomes generate peptide fragments in the cytosol.
• Peptide fragments are transported into the ER from the cytosol via TAP (Transporter Associated with Antigen Processing).
• In the ER, peptides are loaded onto nascent MHC class I molecules.

TAP: Transport of Peptides into the ER

• The transporter complex named TAP moves generating peptide fragments from the cytosol into the ER lumen where they can bind MHC I.
• This step is essential for creating peptide–MHC I complexes destined for the cell surface.

Assembly of Peptide–MHC Class I Complexes in the ER

• Peptide fragments leaking into the ER mix with host proteins encoded by MHC (HLA in humans).
• MHC class I molecules (HLA) are assembled with peptide in the ER before being transported to the cell surface.
• Mouse equivalent: H-2.

MHC Class I: Polymorphism and Genomic Basis

• Human MHC (HLA) chromosome 6 contains class I genes with thousands of polymorphic structures due to allelic variation.
• The map of the human MHC shows multiple class I isoforms; there are 3 class I isoforms per chromosome, leading to a maximum of 6 different class I MHC molecules in an individual (diploid: 3 from each chromosome).
• There is a very low frequency of recombination among these loci.
• MHC class I genes are codominantly expressed on all nucleated cells.

MHC Class I Structure: Heavy Chain and β2-Microglobulin

• Structural components:
  • Heavy chain with α1, α2, α3 domains
  • β2-microglobulin auxiliary light chain
  • Transmembrane segment and cytoplasmic tail
• The external domain forms the peptide-binding site; the heavy chain contributes α1 and α2 to form the binding groove; α3 interacts with CD8 and the TCR less directly.
• The peptide-binding site is a groove formed by the α1 and α2 domains.

Notable Contributors to MHC Class I Structure and Function

• Classic MHC I fold with a1 and a2 forming the peptide-binding groove; α3 and β2-microglobulin provide structural support.
• MHC Class I molecules present endogenous peptides to CD8+ T cells, enabling detection of intracellular pathogens.
• References to structural illustrations credit researchers (e.g., Pam Bjorkman’s group) for the class I fold and general architecture.

Peptide Binding Site: Architecture and Specificity

• The peptide-binding site consists of a groove with a bottom and walls where the peptide sits.
• The binding specificity of Class I MHC is defined by anchor residues of the peptide and by polymorphic residues in the MHC groove.

Determinants of Peptide Binding: Anchor Residues and Peptide Length

• Peptides that bind Class I MHC are generally 8–10 amino acids in length: $8-10$ amino acids.
• Anchor residues are contact points with polymorphic MHC residues that secure the peptide in the groove.
• The T cell receptor (TCR) interacts with both the MHC molecule’s α-helical walls and the bound peptide.
• This interaction underlies MHC-restricted recognition by T cells.

Antigen Presentation and TCR Recognition

• TCRs recognize a composite surface: the MHC molecule and the presented peptide.
• The interaction supports MHC-restricted recognition: TCR binds to the combined complex of MHC plus peptide rather than to either component alone.

Signal 1: Antigen-Specific TCR Activation

• The binding of the peptide–MHC complex to the TCR provides Signal 1 for T cell activation in a two-signal process.
• Co-receptors (CD8 for Class I MHC) participate in stabilization and specificity of the response.

CD8 Co-Receptor: Function and Expression

• CD8 co-receptor provides stabilization of TCR binding to the MHC–peptide complex and helps select T cells with appropriate effector functions for the infection type (e.g., cytolysis).
• CD8 is expressed on approximately $30-40\%$ of T cells in the blood.
• CD8 binds to conserved, non-polymorphic regions of Class I MHC molecules, aiding in proper recognition and signaling.

Co-Stimulation: Signal 2 and T Cell Activation

• Activation requires a second signal (Signal 2) delivered by co-stimulatory interactions:
  • APCs express B7 (CD80/CD86).
  • T cells express CD28.
  • Binding of B7 to CD28 triggers intracellular signaling pathways, leading to gene activation and full T cell activation.
• This co-stimulation ensures that T cells are activated only when APCs indicate a genuine, potentially dangerous infection.
• In the absence of Signal 2, T cells become anergic rather than activated, highlighting the necessity of co-stimulation for productive responses.
• Only professional APCs express both class I MHC and co-stimulatory molecules required for Signal 2.

Growth and Differentiation Signals: Interleukin-2 (IL-2)

• IL-2 is the main T cell growth factor.
• IL-2 provides:
  • Signals to maintain T cell growth
  • Signals to steer and fine-tune effector functions
• Growth and differentiation signals are delivered to CD8+ T cells via cytokines such as IL-2 during activation and expansion.

CD8 T Cell Effector Function: Primary Goal

• Primary goal of CD8+ T cells: Lyse host cells infected with microbial pathogens.
• CD8+ T cells are often activated in responses to viral infections, though some intracellular bacteria may also be targets.

Mechanisms of CTL-Mediated Killing: Inducing Target Cell Death

• CTLs destroy target cells primarily by triggering apoptosis via multiple pathways: 1) Granzymes delivered into the target cell 2) Perforin-mediated pore formation 3) Fas ligand (FasL) engagement of Fas on the target cell, activating caspases
  • Result: Target cell apoptosis and removal of the pathogen-containing cell
• Schematic flow: CTL → delivers granzymes and perforin; FasL-Fas interaction also activates caspases, leading to apoptosis of the infected cell.

Natural Killer (NK) Cells: A Close Relative of CTLs

• NK cells share functional similarities with CTLs but are not MHC-restricted.
• Characteristics:
  • CD3−, CD56+ (and IFN-γ–producing in many contexts)
  • Large Granular Lymphocytes (LGLs) in older terminology
  • Circulate in a state of partial activation, part of the innate immune response
• NK cell receptors include a balance of activating and inhibitory signals to determine whether a target cell is killed.

NK Cells and the MHC I Interaction

• Inhibitory receptors on NK cells recognize MHC class I molecules on healthy (uninfected) cells, delivering a signal to avoid killing.
• When cells down-modulate MHC class I (as some virus-infected cells do), NK cells can be activated via activating receptors to kill the abnormal cell.

NK Cells: Two Key Effector Functions

• Cytolysis: Kill target cells directly
• Cytokine production: Release IFN-γ to shape the immune response and activate other cells
• NK cells are considered a first line of defense against viral infections due to their ability to act before antigen-specific T cells are primed.

Summary: CTL Functions and MHC Restriction

• CTLs express CD8 and are programmed to recognize antigen presented by Class I MHC.
• CTLs are activated by antigen complexed to Class I MHC (signal 1) in the presence of co-stimulation (signal 2) for full activation.
• CD8 co-receptor stabilizes the TCR–MHC–peptide interaction and biases activation toward cytolytic effector functions.
• The antigen presentation pathway for endogenous (intracellular) antigens relies on ubiquitination, proteasomal degradation, TAP transport, and loading onto MHC class I in the ER, followed by surface display and recognition by CD8+ T cells.

Connections to Broader Immunology Concepts

• The two-signal model (Signal 1 via TCR–peptide–MHC and Signal 2 via B7–CD28) helps explain how immune responses are regulated to avoid autoimmunity.
• MHC polymorphism underpins why individuals present diverse peptide repertoires and have varying susceptibility to infections.
• Cross-talk between innate (NK) and adaptive (T cells) immunity shapes the early vs late containment of infections.

Practical Implications and Real-World Relevance

• Understanding endogenous antigen processing is crucial for vaccine design, cancer immunotherapy, and treatments that aim to boost CTL responses.
• Therapies that enhance co-stimulatory signaling or IL-2 signaling can amplify CD8+ T cell responses against intracellular pathogens or tumors.
• NK cell activity is an important consideration in antiviral therapies, transplant biology (graft-versus-host considerations), and immune surveillance.

Key Terminology Recap

• Antigen Presenting Cell (APC)
• Major Histocompatibility Complex (MHC) / Human Leukocyte Antigens (HLA)
• MHC Class I and Class II
• CD8 Co-Receptor
• T Cell Receptor (TCR)
• Signal 1 and Signal 2 in T Cell Activation
• TAP (Transporter Associated with Antigen Processing)
• Endogenous Antigen Processing Pathway
• Ubiquitination and Proteasome
• Endoplasmic Reticulum (ER)
• Anchor Residues
• Peptide Length for Class I Binding ($8-10$ amino acids)
• Granzymes, Perforin, Fas/FasL, Caspases (apoptosis pathways)
• NK Cells and their Activating/Inhibitory Receptors

Quick Reference: Numerical and Quantitative Details

• Peptide length for Class I MHC binding: $8-10$ amino acids
• CD8+ T cells represent approximately 30-40 ext{%} of T cells in blood
• Maximum distinct Class I MHC molecules in an individual: $6$ (3 per chromosome)
• Antigen presentation involves a two-signal model: Signal 1 (antigen–MHC–TCR) and Signal 2 (co-stimulation via B7–CD28)
• Class I MHC interactions primarily present endogenous peptides to CD8+ T cells, enabling MHC-restricted recognition

References to Figures and Conceptual Flow (as described in slides)

• APC–T cell synapse and cytokine signaling shape T cell activation and differentiation
• Endogenous antigen processing: cytosolic proteins → ubiquitination → proteasome → peptides to ER via TAP → loaded onto MHC I in ER → surface expression
• TCR binds to peptide–MHC complex; anchor residues determine binding strength and specificity; TCR recognizes both MHC and peptide
• CD8 stabilizes TCR interaction; Signal 2 (co-stimulation) is required for productive activation; absence leads to anergy
• IL-2 drives T cell growth and differentiation; CTLs execute cytolysis of infected cells via perforin/granzyme and Fas–FasL pathways
• NK cells function as a rapid, innate-like defense, particularly when MHC I is downregulated by pathogens