# Lecture 15: T Cell Activation and Co-stimulation
## Immunology
---
## Learning Objectives
By the end of this lecture, students will be able to:
1. Describe the three signals required for full T cell activation
2. Explain the role of the TCR complex and co-receptors (CD4, CD8) in antigen recognition
3. Describe the major co-stimulatory and co-inhibitory pathways
4. Outline the key signaling cascades downstream of TCR engagement
5. Explain the consequences of T cell activation without co-stimulation (anergy)
---
## Lecture Content
### I. The Three-Signal Model of T Cell Activation
Full T cell activation requires the convergence of three distinct signals. **Signal 1 (antigen recognition)** is provided when the TCR binds a peptide-MHC complex on the surface of an antigen-presenting cell. **Signal 2 (co-stimulation)** occurs when co-stimulatory molecules on the APC engage their receptors on the T cell. **Signal 3 (cytokine signaling)** is delivered by cytokines from the APC and surrounding environment that direct T cell differentiation into specific effector subsets. Signal 1 alone, without the co-stimulatory support of Signal 2, drives the T cell into a state of **anergy** -- functional unresponsiveness to subsequent stimulation. Only when all three signals are provided together does the T cell undergo full activation, proliferation, and differentiation into an effector cell.
### II. Signal 1: TCR-Peptide/MHC Interaction
The **TCR** is a heterodimer consisting of alpha and beta chains in the vast majority of T cells (approximately 95 percent bear alpha-beta TCRs), while a smaller population uses gamma and delta chains. Each chain has a variable (V) domain and a constant (C) domain. The complementarity-determining regions (CDRs), especially CDR3, make direct contact with the peptide-MHC complex: CDR1 and CDR2 of the alpha-beta TCR contact the MHC helices, while CDR3 contacts the peptide itself.
The TCR itself has no intrinsic signaling capacity and relies on the **CD3 complex** to transduce activation signals. The TCR associates with CD3-gamma, CD3-delta, and CD3-epsilon chains (each containing one ITAM) and a CD3-zeta-zeta homodimer (each zeta chain containing three ITAMs), for a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs) per TCR-CD3 complex. These ITAMs become phosphorylated upon TCR engagement, initiating the downstream signaling cascade.
The **co-receptors CD4 and CD8** play dual roles: they stabilize the TCR-peptide-MHC interaction and recruit the tyrosine kinase Lck. CD4 binds the beta-2 domain of MHC class II and is expressed on helper T cells, while CD8 (usually an alpha-beta heterodimer) binds the alpha-3 domain of MHC class I and is expressed on cytotoxic T cells. Both co-receptors associate with Lck on their cytoplasmic tails, and by bringing Lck into close proximity with the CD3 ITAMs, they are essential for efficient signaling.
### III. TCR Signaling Cascade
When the TCR engages its cognate peptide-MHC complex and the co-receptor binds, a carefully orchestrated signaling cascade unfolds. **Lck**, associated with CD4 or CD8, phosphorylates the ITAMs on CD3-zeta and CD3-epsilon. Phosphorylated ITAMs then recruit **ZAP-70** (zeta-chain associated protein kinase 70), which is itself phosphorylated and activated by Lck. Activated ZAP-70 phosphorylates the scaffold proteins **LAT** (linker for activation of T cells) and **SLP-76**, which serve as platforms for the assembly of three major downstream signaling pathways.
The **PLC-gamma-1 pathway** is particularly important. PLC-gamma-1 cleaves PIP2 into DAG and IP3. DAG activates PKC-theta, which triggers the NF-kappa-B pathway (promoting survival and cytokine gene expression), and also activates RasGRP, feeding into the Ras-Raf-MEK-ERK (MAPK) cascade that leads to the transcription factor Fos (part of AP-1). IP3, meanwhile, triggers calcium release from the ER, which activates calmodulin and then calcineurin; calcineurin dephosphorylates **NFAT**, allowing it to translocate into the nucleus. The **Ras/MAPK pathway** converges on the AP-1 transcription factor (Fos/Jun), and the **PI3K pathway** activates Akt, promoting cell survival and metabolic reprogramming.
Three key transcription factors are therefore activated: **NFAT** (the target of the immunosuppressive drugs cyclosporine and tacrolimus, which inhibit calcineurin), **NF-kappa-B** (driving survival genes and cytokines), and **AP-1** (promoting proliferation and cytokine expression). All three converge on the **IL-2 promoter**, driving the production of IL-2, which acts in an autocrine and paracrine fashion to stimulate T cell proliferation.
A detailed signaling diagram of the TCR signaling cascade. At the top, a T cell TCR-CD3 complex engages a peptide-MHC complex on an APC, with CD4 (or CD8) binding the MHC molecule and bringing Lck to the ITAMs. Step 1: Lck phosphorylates CD3 zeta ITAMs. Step 2: ZAP-70 is recruited to phospho-ITAMs and activated by Lck. Step 3: ZAP-70 phosphorylates LAT and SLP-76 scaffold proteins. Three pathways branch from LAT/SLP-76: (A) PLCgamma1 → PIP2 cleavage into IP3 (→ Ca2+ release → calcineurin → NFAT nuclear translocation) and DAG (→ PKC-theta → NF-kB; → RasGRP → Ras-MAPK → AP-1). (B) PI3K → Akt → survival. All three transcription factors (NFAT, NF-kB, AP-1) converge on the IL-2 gene promoter in the nucleus, leading to IL-2 transcription. Cyclosporine and tacrolimus are shown blocking calcineurin, preventing NFAT activation.
### IV. Signal 2: Co-stimulation
The principal co-stimulatory pathway is the **CD28-B7 pathway**. CD28 on naive T cells binds B7-1 (CD80) and B7-2 (CD86) on APCs. B7-2 is constitutively expressed at low levels, while B7-1 is upregulated upon APC activation. CD28 signaling enhances IL-2 production by stabilizing IL-2 mRNA, promotes T cell survival by upregulating Bcl-xL, enhances glucose metabolism through mTOR activation, and lowers the threshold of TCR signaling needed for activation. Without CD28 co-stimulation, the T cell becomes **anergic** -- functionally unresponsive to subsequent stimulation.
**CTLA-4 (CD152)** is the major co-inhibitory counterpart to CD28. Although structurally homologous to CD28, CTLA-4 binds B7-1 and B7-2 with much higher affinity. It is upregulated on T cells after activation and is constitutively expressed on Tregs. CTLA-4 suppresses T cell responses by competing with CD28 for B7 binding, delivering inhibitory signals through the recruitment of phosphatases PP2A and SHP-2, and performing trans-endocytosis -- physically capturing and removing B7 molecules from the APC surface. The importance of CTLA-4 is underscored by the fact that CTLA-4 knockout mice develop fatal lymphoproliferation. In clinical oncology, ipilimumab (anti-CTLA-4) is used to block this checkpoint and enhance antitumor immunity.
The **ICOS-ICOSL pathway** provides co-stimulation to activated T cells through the inducible co-stimulator (ICOS), which binds ICOSL on APCs and non-immune cells. This pathway is particularly important for T follicular helper (Tfh) cell function, germinal center reactions, and antibody responses, and it promotes IL-21, IL-4, and IL-10 production.
The **PD-1-PD-L1/PD-L2 pathway** is another major co-inhibitory axis. PD-1, expressed on activated T cells, engages PD-L1 (broadly expressed on many cell types) or PD-L2 (primarily on APCs). PD-1 signaling recruits SHP-2 phosphatase, which dephosphorylates TCR signaling molecules, inhibiting T cell activation and effector function. Chronic PD-1 engagement promotes T cell exhaustion in the settings of chronic infection and cancer, making PD-1 and PD-L1 important therapeutic targets: nivolumab and pembrolizumab block PD-1, while atezolizumab and durvalumab block PD-L1.
Several additional co-stimulatory receptors of the TNF receptor family further modulate T cell responses. **OX40 (CD134)** promotes T cell survival and memory. **4-1BB (CD137)** enhances T cell proliferation and survival and is incorporated into CAR-T cell constructs to improve persistence. The **CD40L (CD154)-CD40 interaction** between T cells and APCs provides bidirectional activation and is essential for isotype switching and DC licensing.
### V. Signal 3: Cytokine-Directed Differentiation
The cytokines produced by APCs and the surrounding microenvironment during T cell activation determine which effector subset the T cell will become. IL-12 combined with IFN-gamma drives **Th1** differentiation (targeting intracellular pathogens). IL-4 drives **Th2** differentiation (addressing parasites and allergy). TGF-beta combined with IL-6 promotes **Th17** development (for extracellular bacteria and fungi), while TGF-beta combined with IL-2 favors **Treg** induction (for immune regulation). IL-6 with IL-21 supports **Tfh** cell development (providing B cell help in germinal centers). These subset-specific differentiation pathways are detailed further in Lecture 16.
### VI. The Immunological Synapse
When a T cell engages an APC, the interface between the two cells becomes organized into a highly structured contact zone known as the immunological synapse. The **central supramolecular activation cluster (cSMAC)** contains the TCR-peptide-MHC complexes, CD28-B7 pairs, and PKC-theta. Surrounding it, the **peripheral SMAC (pSMAC)** forms an adhesion ring composed of LFA-1 bound to ICAM-1. The outermost **distal SMAC (dSMAC)** contains CD45 (a tyrosine phosphatase) and is enriched in actin. Sustained signaling through this synapse for several hours is required for full T cell activation. The synapse also allows directed secretion of cytokines and, in the case of CTLs, lytic granules, precisely toward the APC or target cell.
### VII. T Cell Anergy
Anergy results when a T cell receives Signal 1 (TCR engagement) without Signal 2 (co-stimulation). Anergic T cells remain alive but are functionally unresponsive. At the molecular level, anergy is maintained by a failure to produce IL-2, upregulation of E3 ubiquitin ligases (GRAIL, Cbl-b, and Itch) that degrade key signaling molecules, and epigenetic silencing of the IL-2 locus. Anergy serves as an important peripheral tolerance mechanism, preventing autoimmunity when self-antigens are presented by non-professional APCs that lack B7 co-stimulatory molecules.
A diagram of the immunological synapse between a T cell and an APC, viewed both from the side and from the T cell face (en face view). Side view: T cell and APC are closely apposed with a narrow cleft. Molecular interactions are shown: TCR-pMHC and CD28-B7 in the center, LFA-1-ICAM-1 forming a ring, CD4 or CD8 bridging to MHC. En face view (looking at the T cell contact surface): concentric ring structure -- central SMAC (cSMAC, yellow) containing clustered TCR-CD3, CD28, PKC-theta; peripheral SMAC (pSMAC, green ring) containing LFA-1-ICAM-1 adhesion molecules and talin; distal SMAC (dSMAC, outer red ring) containing CD45 phosphatase and F-actin. Arrows indicate directed cytokine secretion through the synapse toward the APC.
---