Premed · Premed · Immunology
Lecture 17: Cytotoxic T Lymphocytes and Cell-Mediated Immunity
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the activation and differentiation of CD8+ cytotoxic T lymphocytes (CTLs)
- Explain the perforin-granzyme and Fas-FasL killing mechanisms
- Describe how CTLs achieve targeted killing while sparing bystander cells
- Discuss the role of CTLs in antiviral immunity and tumor rejection
- Explain CD8+ T cell memory and exhaustion
Lecture Content
I. Activation of Naive CD8+ T Cells
Naive CD8+ T cells require activation by professional antigen-presenting cells, primarily dendritic cells. Like CD4+ T cells, they depend on three signals: Signal 1 is provided by TCR recognition of peptide-MHC class I on a DC (with cross-presentation enabling responses against exogenous antigens), Signal 2 comes from CD28-B7 co-stimulation, and Signal 3 involves cytokines such as IL-12, type I interferons (IFN-alpha/beta), and IL-2.
CD4+ T cell help is often required for optimal CD8+ T cell responses. Th1 cells "license" dendritic cells by engaging CD40L on the Th1 cell with CD40 on the DC. Licensed DCs upregulate B7, produce IL-12, and provide superior co-stimulation to CD8+ T cells. Without this CD4+ help, CD8+ T cells may still be activated, but they generate poor memory responses. Strong innate signals, particularly high levels of type I interferon, can sometimes substitute for CD4+ help.
Once activated, CD8+ T cells undergo massive clonal expansion, with a single cell generating more than 10,000 effector CTLs over 7 to 10 days. IL-2, provided both by CD4+ T cells and by the CD8+ T cells themselves in an autocrine fashion, drives this proliferation. As effector CTLs differentiate, they downregulate lymph node homing receptors (CCR7, CD62L) and upregulate tissue-homing molecules, enabling their migration to sites of infection.
II. CTL Effector Mechanisms
CTLs employ two principal killing mechanisms, both of which induce apoptosis in target cells.
The perforin-granzyme pathway is the primary killing mechanism. CTLs carry preformed lytic granules -- specialized secretory lysosomes containing perforin (a calcium-dependent pore-forming protein homologous to complement C9), granzymes (serine proteases, with granzyme A and granzyme B being the most important), and granulysin (an antimicrobial peptide that also induces apoptosis). The killing process begins when the CTL recognizes its target through TCR-peptide-MHC class I interaction and forms an immunological synapse. The microtubule-organizing center (MTOC) reorients toward the synapse, and lytic granules polarize and release their contents through directed exocytosis into the synaptic cleft. Perforin polymerizes and inserts into the target cell membrane, forming pores. Granzymes enter the target cell through these pores and also via mannose-6-phosphate receptor-mediated endocytosis. Granzyme B cleaves and activates caspase-3, initiating the caspase cascade that leads to apoptosis. It also cleaves Bid to generate truncated Bid (tBid), which causes mitochondrial outer membrane permeabilization and cytochrome c release, activating the intrinsic apoptosis pathway. Granzyme A activates a caspase-independent cell death pathway characterized by single-stranded DNA nicks. CTLs protect themselves from their own perforin through cathepsin B on their cell membrane (which inactivates misdirected perforin), protective lipids in the granule membrane, and the directed secretion of granule contents into the synapse, away from the CTL surface.
The Fas-FasL pathway (death receptor pathway) provides a second killing mechanism. Activated CTLs upregulate FasL (CD95L/CD178) on their surface, which binds Fas (CD95) on target cells. Fas trimerization recruits FADD (Fas-associated death domain) and procaspase-8, forming the death-inducing signaling complex (DISC). Caspase-8 activation leads to caspase-3 activation and apoptosis. This pathway is particularly important for eliminating activated lymphocytes during the homeostatic contraction phase and for maintaining immune privilege in sites such as the eye and testis, which express FasL. Autoimmune lymphoproliferative syndrome (ALPS), caused by mutations in Fas or FasL, demonstrates the consequences of failed lymphocyte apoptosis: patients develop lymphadenopathy, splenomegaly, and autoimmunity.
<image>A detailed diagram of CTL killing mechanisms. Panel A (Perforin-granzyme pathway): A CTL forms an immunological synapse with a virus-infected target cell. The MTOC and lytic granules in the CTL reorient toward the synapse. Perforin and granzymes are released into the synaptic cleft via directed exocytosis. Perforin inserts into the target cell membrane forming pores. Granzyme B enters the target cell and activates caspase-3 directly and also cleaves Bid to tBid, which causes mitochondrial cytochrome c release. Both pathways converge on caspase activation and apoptosis (nuclear condensation, DNA fragmentation, membrane blebbing). Panel B (Fas-FasL pathway): FasL on the CTL surface engages Fas on the target cell. Fas trimerizes and recruits FADD and procaspase-8, forming the DISC. Active caspase-8 activates caspase-3 → apoptosis. An inset shows the CTL detaching from the dying cell and moving to a new target (serial killing).</image>
III. Features of CTL-Mediated Killing
Several features make CTL-mediated killing both efficient and precise. CTLs are capable of serial killing: after destroying one target, a CTL detaches and engages another, regenerating its lytic granules between kills. A single CTL can kill upward of 20 targets in succession. The killing is highly specific and precise -- only cells displaying the cognate peptide-MHC class I complex are killed, while bystander cells are spared. This precision is achieved through directed secretion at the immunological synapse and the tight seal that prevents leakage of lytic molecules. Killing is also remarkably fast, taking only about 5 to 10 minutes per target cell. Because CTL-mediated killing induces apoptosis rather than necrosis, the dying cells are cleared by macrophages without releasing inflammatory contents, minimizing collateral tissue damage.
IV. Role of CTLs in Antiviral Immunity
CTLs are the primary adaptive immune mechanism for eliminating virus-infected cells. Viral proteins synthesized within infected cells are processed through the endogenous MHC class I pathway, generating virus-derived peptides that flag infected cells for CTL-mediated destruction. The kinetics of the CTL response follow a characteristic pattern: the primary response peaks at day 7 to 10 post-infection, followed by a contraction phase in which approximately 90 to 95 percent of effector CTLs die by apoptosis, with the remaining 5 to 10 percent surviving as long-lived memory CD8+ T cells. The critical importance of CTLs in viral control is demonstrated by the correlation between HIV progression and the decline in HIV-specific CD8+ T cells, by the role of CTL surveillance in controlling CMV and EBV latency, and by the reactivation of latent viruses (CMV, EBV, JC virus) that occurs during immunosuppression.
V. Role of CTLs in Tumor Immunity
CTLs are key effectors in antitumor immunity, part of the broader concept of immune surveillance. Tumor-associated antigens and neoantigens presented on MHC class I can be recognized by tumor-specific CTLs. However, tumors frequently evade CTL-mediated killing by downregulating MHC class I expression, upregulating PD-L1 to engage PD-1 and drive CTL exhaustion, creating an immunosuppressive microenvironment rich in TGF-beta, IL-10, and Tregs, or losing antigen expression through immunoediting. Checkpoint inhibitors (anti-PD-1, anti-CTLA-4) work by reinvigorating CTL responses against tumors, and CAR-T cell therapy represents an approach in which CD8+ T cells are engineered to target specific tumor antigens.
VI. CD8+ T Cell Memory
After the contraction phase, surviving memory CD8+ T cells persist for the long term and can be divided into several subsets. Central memory T cells (Tcm) express CD62L and CCR7 (lymph node homing markers), reside in secondary lymphoid organs, have high proliferative capacity, and produce IL-2. Effector memory T cells (Tem) lack CD62L and CCR7 (enabling tissue homing), circulate through peripheral tissues, and provide rapid effector function by producing IFN-gamma and granzymes. Tissue-resident memory T cells (Trm) express CD69 and CD103, do not recirculate, and remain stationed in barrier tissues such as the skin, lungs, gut, and reproductive tract, where they provide rapid local protection. Stem cell memory T cells (Tscm) express CD45RA, CD62L, and CD95, possess the greatest self-renewal capacity, and can give rise to all other memory subsets.
Memory CD8+ T cells respond faster and more vigorously upon antigen re-encounter, require less co-stimulation for activation, and are maintained in a state of readiness by the homeostatic cytokines IL-7 (for survival) and IL-15 (for homeostatic proliferation).
VII. T Cell Exhaustion
In settings of chronic antigen exposure, such as persistent viral infections and cancer, CD8+ T cells progressively lose their effector functions -- a state known as exhaustion. Effector functions are lost in a hierarchical fashion: IL-2 production is lost first, then TNF-alpha, then IFN-gamma, and finally cytotoxic capacity. Exhausted T cells express sustained high levels of multiple inhibitory receptors, including PD-1, LAG-3, TIM-3, TIGIT, and CTLA-4. They show reduced proliferative capacity, a distinct transcriptomic and epigenetic profile (with the transcription factor TOX serving as a key driver), and altered metabolism with reduced mitochondrial fitness.
The reversibility of exhaustion is partial. PD-1 blockade can reinvigorate exhausted T cells, forming the basis for checkpoint immunotherapy, but epigenetic changes may limit full functional restoration -- a phenomenon sometimes described as "exhaustion scars." The progenitor exhausted subset, characterized by TCF-1 expression and intermediate PD-1 levels, is the population most responsive to checkpoint blockade.
<image>A graph illustrating CD8+ T cell response kinetics and memory formation. The x-axis represents time (days to months after infection). The y-axis represents the number of antigen-specific CD8+ T cells. The primary response shows: activation phase (days 0-3), expansion phase (days 3-7, exponential increase), peak effector response (day 7-10), contraction phase (days 10-30, ~90-95% die), and stable memory phase (months to years, ~5-10% survive as memory cells). Below the curve, bar charts show the predominant functions at each phase: effector phase (high IFN-gamma, granzyme B, perforin), memory phase (rapid recall, IL-7R and IL-15R expression). An inset compares primary vs. secondary response: the secondary response is faster (peaks by day 3-5), larger magnitude, and longer-lasting. A second inset shows exhaustion: in chronic infection, the effector response does not contract normally but instead progressively loses function, with PD-1, TIM-3, and LAG-3 upregulation indicated.</image>
